Lab Protocols

Acid-Fast Stain Protocols - Ziehl-Neelsen, Kinyoun, Truant Methods

2020-08-22T23:43:00.020+05:30

There are three common acid-fast staining methods, Ziehl-Neelsen (hot), Kinyoun (cold), and Auramine-Rhodamine Fluorochrome (Truant method). The Ziehl-Neelsen method has endured as a reliable and effective way to demonstrate the acid-fast bacteria.

In 1882 Robert Koch reported the discovery of the tubercle bacillus (4) and described the appearance of the bacilli resulting from a complex staining procedure. During the same time period several other researchers (Ehrlich, Ziehl, Rindfleisch, and Neelsen), intending to improve on Koch’s method, introduced modifications to the reagents and the procedure. Franz Ziehl was the first to use carbolic acid (phenol) as the mordant. Friedrich Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin (first used by Ehrlich in 1882). This method became known as the Ziehl-Neelsen method in the early to mid 1890s. In this method heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells. The use of heat in this method has been the reason that this technique is called the “hot staining” method.

Ziehl-Neelsen method	Kinyoun method	Truant method
Carbolfuchsin stain: •Basic fuchsin, 0.3 g •Ethanol, 95% (vol/vol), 10 ml •Phenol, heat-melted crystals, 5 ml •Distilled water, 95 ml •Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water. •Mix and let stand for several days. Filter before use. Decolorizing solvent: •Ethanol, 95% (vol/vol), 97 ml •Hydrochloric acid (concentrated), 3 ml Counterstain: •Methylene blue chloride, 0.3 g •Distilled water, 100 ml	Kinyoun carbolfuchsin solution: •Solution A. Dissolve 4 g of basic fuchsin in 20 ml of ethyl alcohol. •Solution B. Dissolve 8 g of phenol (melted) in 100 ml of distilled water. •Mix solutions A and B together and allow to stand for a few days. Acid-alcohol decolorizing agent: •Ethanol, 95% (vol/vol), 97 ml •Hydrochloric acid (concentrated), 3 ml Methylene blue counterstain: •Methylene blue chloride, 0.3 g •Distilled water, 100 ml •Dissolve by shaking.	Fluorescent staining reagent: •Auramine O, CI 41000, 1.50 g •Rhodamine B, CI 749, 0.75 g •Glycerol, 75 ml •Phenol (heat melted crystals), 10 ml •Distilled water, 50 ml •Mix the two dyes well with 25 ml of the water and the phenol. Add the remaining water and glycerol and mix again. •Filter the resulting staining fluorescent reagent through glass wool and store at 4 oC or room temperature. Decolorizing solvent: •Ethanol, 70% (vol/vol), 99.5 ml •Hydrochloric acid (concentrated), 0.5 ml Counterstain: •Potassium permanganate, 0.5 g •Distilled water, 99.5 g

Basic Smear Preparation

Clean a glass slide (some labs will provide pre-cleaned slides; be sure to remove any dust or crushed glass debris) according to instructions provided by your instructor.
Prepare the sample according to instructions provided by your instructor. Make certain that an aerosol is not generated during this process.
Using a sterile pipet or microbiological loop, apply a small sample of the specimen to the slide by slowly spreading the liquid to make a thin film; If you are using solid matter from a colony, be sure to choose a very minute sample and spread it into a very thin film. Applying the cells before adding water (or other mixing fluid) will help the cells adhere to the slide. The size of the film should be about 1 cm in diameter. Avoid any actions that would splatter droplets of the sample in the surrounding area.
Allow the smear to dry completely.
Fix the smear at 80 oC for 15 minutes or for 2 hours on a hot plate set for 65 oC to 70 oC.
Proceed to the staining protocol of your choice.

Ziehl-Neelsen method for acid-fast staining

Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
Place a slide with an air-dried and heat-fixed smear on suitable staining device. Cut a piece of absorbent paper to fit the slide and saturate the paper with the carbolfuchsin stain.
Carefully heat the underside of the slide by passing a flame under the rack or by placing the slide on a hot plate until steam rises (without boiling!). Keep the preparation moist with stain and steaming for 5 minutes, repeating the heating as needed.
(CAUTION: overheating causes spattering of the stain and may crack the slide).
Wash the film in a gentle and indirect stream of tap water* until no color appears in the effluent.
Holding the slide with forceps, wash the slide with the decolorizing solvent. Immediately wash with tap water*, as above. Repeat the decolorizing and the washing until the stained smear appears faintly pink and the fluid washing off the slide runs clear.
Flood the smear with the methylene blue counterstain for 20 to 30 seconds, and wash with tap water*, as above.
Gently blot, or air dry the smear.
Examine under oil immersion.** Acid-fast bacteria appear red, and non-acid-fast bacteria (and other organisms and cellular materials) appear blue.

* Most labs use deionized or distilled water for all lab procedures. See note in Tips and Comments section regarding the use of tap water.
** For clinical samples, examination of at least 300 fields is required before declaring a specimen negative for acid-fast bacteria.

Kinyoun method for acid-fast staining

Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
Flood slides with Kinyoun’s carbolfuchsin reagent and allow to stain for 5 minutes at room temperature.
Rinse with deionized water and tilt slide to drain.
Decolorize with acid-alcohol for 3 minutes and rinse again with deionized water.*
Redecolorize with acid-alcohol for 1-2 minutes or until no more red color runs from the smear.*
Rinse with deionized water and drain standing water from the slide surface by tipping the slide.
Flood slide with methylene blue counterstain and allow to stain for 4 minutes.
Rinse with distilled water and allow to air dry.
Examine under high dry (400X) magnification, and confirm acid-fast structures under oil immersion (1000X).

*Most practitioners apply decolorizer until the fluid washing off the slide runs clear.

Truant method for acid-fast staining

Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
Wash the slide with a gentle and indirect stream of distilled water until no color appears in the effluent.
Flood the smear with the decolorizing agent for 2 to 3 minutes, and then wash with distilled water as above.
Flood the smear with the permanganate counterstain for 2 to 4 minutes.
Wash the slide with distilled water as above, blot with absorbent paper, and dry.
Examine the slide with a fluorescence microscope equipped with a BG-12 exciter filter and an OG-1barrier filter. Acid-fast bacteria appear as brightly fluorescent, yellow-orange cells in a dark field; non-acid-fast cells appear dark.

Zebrafish Care and Experimental Techniques: In Situ Hybridization - Zebrafish Sections

2011-11-06T01:19:00.001+05:30

**Day 0:

Goals: To preserve zebrafish and obtain sections using the cryostat.
**Everything used on this day needs to be RNase free. Wear gloves and be sure to use only PBS that has been treated with DEPC.

1. Anesthetize zebrafish

2. Fix in freshly thawed BT fix for 3 hours at room temperature. Keep solution on the "belly dancer" so that it is constantly moving.

3. Rinse 3 x 5 minutes in 1X PBS. Leave embryos in pbs until you are ready to section them. Embryos can be stored in the fridge until sectioning, but keep the days between fixing, sectioning and staining as short as possible.

4. Rinse fish in 30% sucrose and PBS until embryos sink approx. 30mins.

5. Trnsfer embryos from sucrose to plastic molds, 5 fish pre mold, with heads facing the side with writing. Absorb as much sucrose as possible.

6. Add OCT to the mold, and swirl fish until they are oriented correctly. Freeze in -80 degree freezer.

7. Follow instructions for sectioning using the cyrostat.

**Day 1:

Goals: Permeabilize zebrafish tissue, block non-specific binding, and bind specific probes (GFAP and slitrk probes)

**Note: Everything used on this day needs to be RNAse free!!! Wear gloves and use special tips. Keep Day 1 glassware separate from all other glassware. There are three Coplin jars labeled Day 1 – one is labeled for TEA and the other two are used for PBST rinses. Keep them labeled and cover with parafilm when not in use and only use for Day 1.

Steps 1-9 should be performed in the hood. All wash steps are performed in Coplin jars. Each jar holds about 50 mL of liquid.

1. Thaw sections for 15 minutes at Room Temperature

2. Fix in BT Fix for 10 minutes in hood. You will want to take a tube of BT Fix out of the freezer and place in fix fridge to thaw the day before you start this protocol. Overlay slides with about 1 mL of fix. Might have to use pipette tip gently spread fix so that it covers the whole slide. While slides are fixing mix PBST.

3. Wash 2 x 5 minutes in PBST to rinse off fix from previous step. While slides are rinsing mix proK.

4. Add Proteinase K (ProK) 10 ug/mL for 10 minutes to permeabilize the tissue. Overlay slides with about 0.5 mL ProK. The timing on this step is critical, so do not let it go over time. If you treat with ProK too long the cells can disintegrate or all the RNA could go out of them.

5. Wash 2 x 5’ in 1X PBST to rinse off ProK
6. Fix again in BT fix 10 minutes (in hood). Overlay slides with about 0.5 mL. This is to preserve the tissue in the permeabilized state.

7. Wash 2 x 5 minutes in 1X PBST. During this step, pre-warm the hybridization oven and HM (hybridization media) in dry bath incubator 50 C and mix TEA.

8. Acetylation for 10 minutes in a Coplin jar (pre-mix by adding 125 ul acetic anhydride (located in the “Flammable” cabinet under the hood) to 50 mls 0.1 M TEA. Mix in a 50 mL conical tube in hood.) This step eliminates the charge on the superfrost plus slides.

9. Wash 2 x 5 minutes in 1X PBST

10. Pre-hybe with HM for 2 hours in 50 C hybridization oven to block non-specific binding. Overlay each slide with 400 μl HM.

11. Add fresh HM + probe to the slides and keep in 50 C oven overnight (1:1000 dilution). Use the GFAP probe as a positive control and then use other slitrk probes on the remaining slides depending on the experiment. During this step the slitrk and GFAP probes are binding to the RNA and move PBST from Day 1 bottle into PBST Day 2 and # Bottle.

Day 2:

Goals: Wash off unbound probe from Day 1, block non-specifc binding, and bind antibody to probe that is bound to RNA.

Steps 1-6 performed in Coplin jars.

1. Wash 15 minutes in 2X SSC (dilute from 20X stock) at 50 C (pre-warm solution in water bath; turn water bath on about 20 minutes before needed)

2. Wash 5 minutes in 2X SSC at Room Temperature

3. Add 20 ug/ml RNAse A (dilute in 2X SSC) for 30 minutes at 37 C. Use the hybridization oven for this step and overlay the slides with about 0.5 mL.

4. Wash 5 minutes in 2X SSC at Room Temperature

5. Wash 2 x 30 minutes in 0.2X SSC (dilute from 20X stock) at 50 C (pre-warm solution by placing in water bath ahead of time)

6. Wash 2 x 10 minutes in 1X PBST at Room Temperature

7. Add Blocking Solution (1X PBST and 10% sheep serum) for 1 hour at Room Temperature in the humid chamber. Overlay each slide with about 1 mL of blocking solution.

8. Add Blocking Solution + anti-DIG-AP antibody at 1:5000 overnight at 4 C. Overlay each slide with about 1 mL and keep in humid chamber. Place humid chamber in the fix fridge.

Day 3:

Goals: Wash off unbound antibody from Day 2. Perform reaction to reveal staining pattern in sections.

1. Discard liquid on slides

2. Wash 4 x 15 minutes in 1X PBST. These washes are rinsing off the unbound antibody from Day 2.

3. Wash 2 x 5 minutes in 100 mM Tris pH=9.5 (Dilute from 1 M stock). The tablets in the next step contain tris, so this step is acclimating the tissue to that solution.

4. During step 3 combine 5 ml Millipore H2O and ½ of a NBT-BCIP tablet in a 15 mL conical tube. Shake well to dissolve tablet. Keep in a drawer away from light.

5. Add about 500 μl solution from step 4 to each slide in the humid chamber. Keep at Room Temperature in the dark. Easiest to store humid chamber in a cubby/cabinet with a door.

6. Check slides under microscope at the end of the day for staining pattern. Slides can be left for up to two days before stopping the staining reaction. If you are leaving slides longer than one day, change the staining solution after 24 hours. Rinse slides in 100 mM Tris for 5 minutes then add fresh staining solution to each slide.

7. To stop the staining reaction, wash slides 2 x 5 minutes in 1X PBS. (Insert pictures of successful staining we have seen so far.)

8. Add mounting medium and coverslip. Store in a slide box at 4 C (fix fridge).
See the drop box files for staining worksheet (with recipes). Use a staining worksheet each time you complete a stain, making notes about that particular trial. Put the worksheet in the " In Situ Zebra Fish Protocol Worksheet" binder. If you uncover something significant that is helpful or requires a change in protocol, make note of it on the worksheet in an obvious way.

Zebrafish Care and Experimental Techniques: Real Time PCR

2011-11-06T01:18:00.001+05:30

Materials:
-primers (24 and 25 are for slitrk 3)
-cDNA (40 hour/90 hour)
-10mM dNTP (nucleotides)
-buffer 5x
-water
-enzyme (cooler in freezer)

Combine in a PCR tube (50 microliters of solution):
1. 35.5 uL of water
2. 10uL 5x buffer
2. 1uL of JR 24
3. 1uL of JR 25
4. 1uL of dNTP
5.1uL of cDNA
6. 0.5 uL of Fusion hot start enzyme

Add water to the PCR tube first. Make sure you use the PCR tubes because they fit in the machine.
After all contents have been added, flick PCR tube and centrifuge for 4 seconds.

To Run the PCR Machine:
The power switch is on the back. The lid will click, and then the wheel should be twisted until you feel resistance.
Program: JRRTPCR
enable heated lid
The reaction takes several hours. The machine with have a block temp of 4 degrees when done.
Remember to turn off the machine when finished using it.

Zebrafish Care and Experimental Techniques: Midi-Prep of Plasmid DNA

2011-11-06T01:16:00.002+05:30

Day 1: see "Bacterial Transformation by Heat Shock"

**Day 2: Starter Cultures and Inoculate into Large Bacteria Flasks**

NOTE: Starter culture is preferable, but not required; can immediately go to larger flasks if you want to

STARTER CULTURE:
1. Aliquot 2ml of the LB+Amp solution (0.02g Amp/200ml LB) into 14ml round bottom tubes
2. Using a yellow pipet tip, pick up an isolated large colony and drop the tip into the correct tube
3. Cap and place into incubator (37C) with shaker set to 200rpm for ~6 hours

Note: At this point, can refrigerate starter cultures until ready to inoculate and do midi-prep kit

INOCULATION:
1) Add Amp to LB broth at 100ug/ml (so, for 500ml of LB broth, add .05g Amp)
2) Add 100ml of LB+Amp into a 1L flask for every plasmid you are inoculating
3) Transfer 200ul of bacteria from starter culture into corresponding flask
4) Cover each 1L flask with aluminum foil
5) Incubate for 12-16 hrs in Dana Incubator (if necessary, use paper towels to prevent flasks from shaking too much)

**Day 3: Midi-Prep Kit**

NOTES:
-with new box, centrifuge any liquid in small tube before using it
-add LyseBlue and RNase A to P1 buffer first time kit is opened (all 110ul of each) and store in refrigerator
-store Buffers 1 and 3 in fridge

1) Pour bacteria from 1L flasks into large round, capped bacteria bottles (LB should be cloudy) and store on ice until ready to use
2) Centrifuge in supply room at 6000xg for 15 min (temp at 4C) (use JA-14 rotor); must turn on power before opening centrifuge
-use chart to convert g to rpm (note rotor #)
3) While centrifuging, take 95% ethanol and pour a bit into bacteria flasks and bacteria bottles; let sit for 10 minutes and then pour down the drain
4) Once done centrifuging (should see pellet at bottom), pour off excess liquid into a waste flask (one of the ones used for bacteria)
5) Add 4ml of Buffer P1 (in fridge) to each pellet
6) Vortex to re-suspend all the pellet into the liquid
7) Pour contents into LABELED 50 ml conical tubes
8) add 4ml of Buffer P2, shake vigorously, and let sit for 5 mins; soln should turn blue; this step is lyses the cells open
NOTE: THIS IS THE ONLY TIME SENSITIVE STEP. NO LONGER THAN 5 MINS.
9) While waiting, prepare filter cartridges by putting caps onto filters and labelling them; line filters up next to corresponding conical tubes
10) With 45 seconds left, open caps of conical tubes (should be gooey)--DO NOT MIX UP CAPS
11) After 5 mins, add 4ml of Buffer P3 and mix; blue should disappear; this step neutralizes the bacteria
12) Transfer liquid from conical tube into filter (with cap)and let it sit for 10 minutes
*Conical tubes now become waste containers*
13) While filter is sitting for 10 min, place column (QIAGEN-tip) onto conical tubes using blue holders and drain 4L of QBT buffer through column (equilibrating column)
14) After 10 minutes, plunge liquid from filter into LABELED column; DNA is binding to column
*14b) OPTIONAL: can run the lysate through the column 2x to ensure all the DNA has bound to column; if you do this, the conical tube that catches the liquid MUST be clean
15) Once lysate has gone through column, empty waste in conical tube (dump into waste flask)
16) Wash column 2x with 10ml of QC Buffer (empty waste container between the two washes)
17) Place filters into 14ml round bottom tubes
18) Elute DNA with 5ml of QF buffer into a clean 14 mL round bottom tube.

(Can stop here if necessary and proceed later--keep eluate on ice or @ 4C)

19) Precipitate DNA by adding 3.5ml of room temp isopropanol (IPOH, 2-propanol) and mix in 14ml round bottom tubes
20) Pour liquid into LABELED Beckman ultra clear tubes (will change with new tubes)
21) Centrifuge the Beckman tubes using ultracentrifuge in Dana for 30 min at 4C at >15000xg
-use swinging bucket rotor
22) Discard liquid from Beckman tubes; should see opaque pellets at the bottom of the tube (that's DNA!)
23) Place Beckman tubes upside down on paper towel
24) Place 1ml of 70% ethanol into each B. tube and pipet up and down GENTLY to get all of the DNA off the bottom
25) Put this 1ml soln into a new eppendorff tube (LABELED)
26) Microfuge epp. tubes for 10 min; small pellet of DNA on side should be visible
27) Pipet out excess liquid and lay on its side to let all excess ethanol to evaporate (~10 min); pellet should appear dry but still visible
28) Suspend DNA in 100-150ul of ddH2O
29) Flick to ensure that the DNA has dissolved into the water and store in fridge

NOTE: to clean waste beaker, add a splash of bleach, let sit for 10 minutes, pour down drain, and rinse thoroughly

Zebrafish Care and Experimental Techniques: Gel Electrophoresis

2011-11-06T01:14:00.002+05:30

Materials:
-All materials are EtBr exposed, so wear gloves!
-Materials are kept on a tray on the lab bench
1. TAE solution
2. Agarose
3. EtBr
4. Plate set-up
5. DNA ladder (in fridge)
6. PCR product

For 70mls of 1% gel solution:
1. Add 70 mls of 1X TAE to the flask
2. Add 0.7g of agarose to the flask
3. Microwave the solution for 1:30 mins, watching for bubbles
4. Microwave again until the solution clears and bubbles but does not overflow
5. Allow the solution to cool to 50 degrees celsius (warm to touch)
5. Under the hood, add 3.5uL of EtBr to the solution (before it cools completely!)
5. Stir the solution gently so that the EtBr dissolves
6. Once dissolved, pour the solution onto the gel plate set-up. Make sure the comb is properly oriented and that the screws are tightened.
7. Allow plate to cool for at least 25 mins
8. Place plate into buffer (TAE) and gently remove the comb
9. Add enough TAE to cover the wells
10. Use a DNA ladder, 20 uL in one well
11. Spin PCR tube
12. Add 10uL of DNA loading buffer to a 50uL PCR tube (the buffer should be at least 1/6th of the total solution volume) to dye and weigh down the solution
13. Add PCR solution to a well
14. Connect the voltage channels, make sure the red (+) and black (-) are oriented correctly. Red should be plugged into red and black should be plugged into black. Red should also be on the bottom. (NOTE: when you put the cover on, press hand on clear top otherwise the entire holding device will flip over)
15. Set the voltage to 80 volts for about 30 mins. There should be bubbles rising gently from the bottom to the top of the liquid.
When viewing the gel, make sure to zoom in so you are taking the closest picture possible. You may also need to to manually expose if the auto expose doesn't show bands well.

Multichannel Images with Bright-field in Nikon Elements

2011-11-06T01:10:00.000+05:30

To take a multichannel image in Elements:
1. click acquire --> capture multi channel image --> manually
2. There should now be an open window with tabs along the bottom for each of the image channels
3. Bright-field should be included in those tabs
4. If Bright-field is not included, you will need to add a channel.
a. click acquire --> capture multichannel image --> multichannel setup
b. click on an unchecked black box to add a channel, assign the channel the appropriate settings
c. for Bright-field set the comp. color to Bright-field
d. when you close re-open the multichannel image you should have a bright-field tab
5. Click the tab along the bottom that corresponds with the image you want to take
6. set-up the image using the microscope or i-control
7. click capture when you are ready to take the picture
8. Take all of the images you need to compile and then click the four box icon in the left hand corner. The icon is a shaded box with three colored boxes popping off if it.
9. You should then see an array of your images, including the composite.
10. you can save the entire array, or individual images. To save the array click save as. To save individual images, click on an image so that it becomes surrounded by a red box, then click save as.

Calcium phosphate transfection protocol - Protocol for cell transfection using the calcium phosphate method

2011-08-04T11:55:00.001+05:30

Procedure:
Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Add 40 μl per well immediately after plating (20 μl each of the luciferase plasmid with 20 μl of the beta gal mix). All mixing (except that which requires vortexing) should be done in the sterile cell culture hood.

Total volume	400 μl	600 μl	800 μl
Sterile 2.5 M CaCl₂	25 μl	37.5 μl	50 μl
Plasmid	10 μg	15 μg	20 μl
Sterile H₂0	Total of 200 μl when added to above.	Bring to total of 300 μl with sterile H₂0	Bring total to 400 μl with H₂0
	Combine the above three reagents in a hood using a sterile 1.5 ml Eppendorf tube. Mix gently. Do not vortex.
Sterile 2X HBS (hepes buffered saline)	Add 200 μl drop-wise with gentle agitation of the above mixture (setting of 2 on vortex mixer)	300 μl added dropwise with gentle agitation. Then invert several times to mix completely.	400 μl added dropwise.

Notes:
After adding the 2X HBS, cap the eppendorf and let stand at room temp for no less than 15 min. When ready to apply the mix to cells, combine equal amounts of the reporter plasmid with beta-gal control mix. Mix gently by inverting several times and add 40 μl of the mix to each well of a 24 well plate (this will require optimization depending on the specific experiment) swirling the cells gently to disperse the transfection mix into the media. For transfection of another plasmid/construct on 10 cm plates, a total volume of 300 μl of transfection mix into 6 ml of media works well.

Dr. Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA

Mouse adipocyte glucose uptake assay - Protocol for glucose uptake assay in mouse adipocytes

2011-08-04T11:53:00.002+05:30

Procedure

Day 0 cell culture:

Grow 3T3-L1 cells in high glucose DMEM + 10% Calf Serum + P/S/F in 6-well plates.
After cell have reached complete confluent, replace growth media with stimulation media containing:

High glucose DMEM:
10% FCS, P/S/F
1 µl / ml of Insulin (10 mg/ml, 1000x in HEPES, final 10 µg/ml)
1 µl / ml IBMX (3-isobutyl-1-methylxanthine, 500 mM stock, 1000x), final 0.5 mM
0.1 µl / ml Dexamethasone (10000X in ethanol, final 1 µM).
Day 3

Replace with fresh media containing Insulin only, no IBMX, no Dexamethasone. Change the media on day 6. Cells will be differentiated on day 7-9.

Day 6
Cells will be cultured in serum-free DMEM for 2 hr ± treatments

Incubate cells in 1 ml of KRH with 100 nM insulin for 20 min ± treatments.
Add 1 µCi of 2-deoxy-D-[2,6-3H]-glucose (50 µl in volume) to each well for 10 min.
Wash cells with KRH buffer 3X.
Lyse cells in 1 ml of RIPA buffer.
Count 300 µl.

KRH buffer:
25 mM HEPES-NaOH [pH 7.4]
120 mM NaCl
5 mM KCl
1.2 mM MgSO₄1.3 mM CaCl2
1.3 mM KH₂PO₄
References
J Clin Invest. 2005, 115, 291-301.

Dr. Guo-Ping Shi
Harvard Medical School
Brigham and Women's Hospital
Cardiovascular Medicine
Boston, MA

Cardiomyocyte immunofluorescence protocol - Protocol for glucose uptake assay in mouse adipocytes

2011-08-04T11:52:00.001+05:30

Procedure describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes

View a pdf version of this page

Anti-sarcomeric – α actinin and ANP Staining

Fix cells as usual in 3.7% paraformaldehyde in PBS.
Rinse cells twice with PBS after fixation.
Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min.
Treat cells with antibodies for α-actinin eg. alpha sarcomeric actin antibody [alpha Sr-1] (ab28052) and ANP diluted in the blocking buffer as above for 1 hour.
Wash x3 in PBS for 5 min each.
Add secondary ab in the same blocking buffer (for green ab: CY2 or fluoroscein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min (though these will require some optimization)
Wash in PBS x3
Add DAPI solution to coverslips for 15 min.
Wash x2 in PBS.
Add mounting solution and coverslip. Image cells within 2 days. The α-actinin stain is stable for quite some time but the ANP stain tends to bleach out within about 1-2 weeks.

Immunostaining for ERα or ERb

Fix cells as usual in 3.7% paraformaldehyde in PBS.
Rinse cells twice with PBS after fixation.
Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min.
Treat cells with antibodies for α-actinin (1/200 dilution) diluted in the blocking buffer as above for 1 hour.
Wash x3 in PBS for 5 min each.
Then treat cells with anti-ERα (or ERb) antibody at optimized dilution overnight at 4°C.
Wash with PBS x3.
Treat with secondary antibodies as above (same dilutions) for 45 min to an hour.
Wash x3 with PBS.
Add DAPI solution for 15 min.
Wash x3 in PBS, then coverslip.

Dr. Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA

Introduction to apoptosis - A brief overview of the mechanisms of apoptosis

2011-08-04T11:50:00.000+05:30

Apoptosis or programmed cell death is defined as "a mechanism of cellular suicide which occurs after sufficient cellular damage".
Apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs and eventually these blebs separate from the dying cell and form "apoptotic bodies". Apoptotic cells also cease to maintain phospholipid asymmetry in the cell membrane, and phosphotidylserine appears on the outer leaflet. The mitochondrial outer membrane also undergoes changes that include loss of its electrochemical gradient, and substances like cytochrome c leak into the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic bodies and the dying cell. The apoptotic cell does not provoke an inflammatory response, and only individual cells are affectedby apoptosis in vivo.

The mechanisms of apoptosis

Apoptosis can be induced in response to many external stimuli (extrinsic pathway) including activation of cell surface receptors such as Fas, TNFR1 (tumor necrosis factor receptor 1), TRAIL-R1 (TNF-related apoptosis inducing ligand receptor 1), TRAIL-R2, p75-NGFR (p75-nerve growth factor receptor) and others. These "death receptors" have two distinct signaling motifs: death domains (DD) and death effector domains (DED) that allow them to interact with other proteins involved in the apoptosis cascade. Typically the extrinsic pathway involves activating the initiator caspase, caspase-8, which in turn either activates caspase-3 or cleaves the Bcl-2 family member, Bid, leading to the formation of the apoptosome and activation of caspase-9.
The alternative mitochondrial pathway (intrinsic pathway) can be activated by events such as DNA damage. Themitochondrial pathway involves members of the Bcl-2 family that regulate cytochrome c release from the mitochondria. Expression of phagocytic recognition molecules at the surface of apoptotic cells may lead at anystage to their removal from the tissue. One of the earliest changes in apoptosis is the movement of negatively charged phospholipids molecules (phosphotidylserine) from the inside of the cell membrane to the outside.
Annexin-V labelling of phosphotidylserine exposure is an established procedure for identifying cells at an earlier stage in apoptosis.
View our apoptosis pathway diagram for further details.

DNA fragmentation analysis protocol - Semi-quantitative method for measuring apoptosis by Richard Pattern (Tufts-New England Medical Center)

2011-08-04T11:49:00.000+05:30

Procedure:

Pellet cells
Lyse cells in 0.5 ml detergent buffer: 10 mm Tris (pH 7.4), 5 mm EDTA, 0.2% Triton
Vortex
Incubate on ice for 30 min.
Centrifuge at 27000 g for 30 minutes
Divide supernatants into 2-250 μl aliquots
Add 50 ul ice cold 5 M NaCl to each followed by vortexing
Precipitate DNA: Add 0.6 ml 100% EtOH and 150 μl 3 M Na acetate, pH 5.2 place at –80oC freezer for 1 hr. Centrifuge 20,000 g for 20 min and pool DNA extracts by re-dissolving by adding 400 μl total) of extraction buffer (10 mM Tris and 5 mm EDTA).
Add 2 μl (10 mg/ml) DNase free RNase. Incubate for 5 hr at 37^oC.
Add 25 μl Proteinase K at 20 mg/ml and 40 μl of buffer (100 mm Tris, pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65^oC.
Extract DNA with phenol, choloform, isoamyl alcohol and precipitate with EtOH.

From Kotamraju et al JBC, 2000 DNA Fragmentation:
A distinctive feature of apoptosis at the biochemical level is DNA fragmentation. This method was used as a semiquantitative method for measuring apoptosis. The culture medium was removed and centrifuged at 3000 × g for 5 min to collect detached cells. Adherent cells were lysed with a hypotonic lysis buffer (10 mM Tris-HCl, pH 8.0) containing EDTA (10 mM) and Triton X-100 (0.5%) and then pooled with pellets made of detached cells. RNA was digested using RNase (0.1 mg/ml at 37 °C for 1 h) followed by proteinase K treatment for 2 h at 50 °C. DNA was extracted with a mixture of phenol, chloroform, and isoamyl alcohol (25:24:1). DNA was precipitated by adding an equal volume of isopropyl alcohol, stored overnight at 20 °C, and centrifuged at 12,000 × g for 15 min at 4 °C. The pellet was air-dried, resuspended in 20 μl tris acetate EDTA buffer supplemented with 2 μl of sample buffer (0.25% bromphenol blue, 30% glyceric acid), and electrophoretically separated on a 2% agarose gel containing 1 μg/ml ethidium bromide and visualized under ultraviolet transillumination.

Dr. Richard Pattern
Tufts-New England Medical Centre
Molecular Cardiology Research Centre
Boston, MA

Caspase detection protocol Procedure for detection and immunostaining of caspases, including list of available kits

2011-08-04T11:47:00.000+05:30

Introduction

Caspases are essential in cells for apoptosis, one of the main types of programmed cell death in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Caspases were first implicated in apoptosis when CED-3, a protein required for programmed cell death in Caenorhabditis elegans, was found to have close homology with the mammalian interleukin-1 -converting enzyme (ICE or caspase 1) and that over-expression of ICE induced apoptosis. Failure of apoptosis is one of the main causes of tumour development and autoimmune diseases. This coupled with the unwanted apoptosis that occurs with ischaemia or Alzheimer's disease, has raised interest in caspases as potential therapeutic targets.
Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. As proteases, they are enzymes that cleave other proteins. They are called cysteine proteases, because they use a cysteine residue to cut those proteins, and called caspases because the cysteine residue cleaves their substrate proteins at the aspartic acid residue. The generic name for all members is caspase with the c denoting a cysteine protease and aspase referring to the aspartate specific cleaving ability of these enzymes. The individual members are then numbered according to their chronological order of publication. To date 14 caspases have been identified (12 human and 2 murine).

Types of caspase proteins

Initiator caspases (e.g. CASP1, CASP2, CASP8, CASP9, CASP10, CASP12) cleave inactive pro-forms of effector caspases, thereby activating them.
Effector caspases (e.g. CASP3, CASP6, CASP7,CASP14) in turn cleave other protein substrates within the cell resulting in the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors.

Immunohistochemical staining protocol

Materials required:

Active Caspase 3 antibody, rabbit pAb (ab2302)
prepared, fixed samples on slides
Triton X-100
PBS
blocking buffer (PBS/0.1% Tween 20 + 5% horse serum)
goat anti-rabbit Cy5 conjugate secondary antibody (ab6564)
mounting medium
humidified chamber

Permeabilize the fixed samples by incubating in PBS/0.1% Triton X-100 for 5 mins at room temperature.
Wash three times in PBS, for 5 mins at room temperature.
Drain the slide and add 200 μl of blocking buffer (PBS/0.1% Tween 20 + 5% horse serum). Use of serum from the host species of the conjugate antibody (or closely related species) is suggested. Lay the slides flat in a humidified chamber and incubate for 1-2 hours at room temperature. Rinse once in PBS.
Add 100 μl of the active Caspase 3 antibody diluted 1/200 in blocking buffer. You can also prepare a slide with no active Caspase 3 as a negative control. Incubate slides in a humidified chamber overnight at 4°C.
The following day, wash the slides three times, 10 mins each in PBS/0.1% Tween 20 at room temperature.
Drain slides and add 100 μl of goat anti-rabbit Cy5 conjugate diluted 1:500 in PBS. Lay the slides flat in a humidified chamber, protected from light, and incubate for 1-2 hours at room temperature. Wash three times in PBS/0.1% Tween 20 for 5 mins, protected from light.
Drain the liquid, mount the slides in a permanent or aqueous mounting medium and observe with a fluorescence microscope.

Abcam have many caspase antibodies and kits available. The above is a suggested protocol only which may require optimization. Customers will need to use the protocols provided when using Caspase kits:

Caspase detection kits:

Apoptosis induction protocols - Procedure for biological and chemical induction of apoptosis in cells

2011-08-04T11:46:00.000+05:30

Apoptosis may be induced in experimental systems through a variety of methods. In general, they can be divided into 2 categories: a) biological induction; and b) chemical induction.

A) Biological induction of apoptosis

Activation of either Fas or TNF-receptors by the respective ligands or by cross-linking with agonist antibody induces apoptosis of Fas- or TNF receptor-bearing cells. Below is general protocol used to induce apoptosis using anti-Fas receptor (CD95) mAb in Jurkat Cells.
1. Grow Jurkat cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified, 5% CO₂ incubator at 37°C.
2. Suspend the cells in fresh medium at a concentration of 1 × 105 cells/ml. After two to three days of incubation in a 37°C, 5% CO₂ incubator, harvest the cells by centrifugation at 300–350 × g for 5 mins.
3. Resuspend cells in fresh medium to 5 × 105 cells/ml and add CD95 mAb to a final concentration of 0.05 – 0.1 μg/ml. Incubate for 3–6 hours in a 37°C incubator. As a negative control, incubate untreated cells (without CD95) under the same conditions. (Stop here for homogeneous assay, or plate the cells in a 96-well plate.)
4. Harvest the cells by centrifugation at 300–350 × g for 5 mins.
5. Remove all medium and resuspend cells in PBS.
6. Repeat centrifugation and resuspend the cell pellet in PBS to 1.5 × 106 cells/ml.
7. Proceed to apoptosis detection.

B) Chemical induction of apoptosis

Depending on the agent selected and the concentrations used, maximal induction of a particular protein may occur within 8 to 72 hours post-treatment. However, not all proteins are affected by reagents in a particular cell line. The following protocol is based on p53-dependent G1-arrest that occurs in response to DNA damage by chemical
agents. A typical time course for p53 induction is 40 to 48 hours treatment with a DNA damaging agent.
1) Inoculate each of 2 or more 10 cm² tissue culture dishes for adherent cells or T-75 flasks for non-adherent cells with approximately 1 x 10⁶ cells. One dish or flask will be used as negative control for non-induced or basal level expression.
2) Confirm that cells are growing by visual inspection of tissue culture dishes or by viable cell counts on non-adherent cells in T-75 flasks. Add DNA damaging agents to recommended final concentrations. The list below gives suggestions of final concentrations that can be used for several well known apoptosis inducing chemicals:
Examples:

0.2 μg/ml Doxorubicin (stock prepared in H2O, 25 μg/ml)
5-Fluorouracil (stock prepared in DMSO)
100-58 nM Paclitaxel (stock prepared in DMSO)
60 nM Vinblastine (stock prepared in methanol)
1 mM staurosporine in DMSO

Add appropriate volume of buffer or solvent (i.e. DMSO) to the non-induced control.
3) Check cells to determine if cells have begun to apoptose. This can be assessed by checking the morphology of the cells (cells will become granulated and blebbing may be observed). Viability can be checked using eg trypan blue cell counting. Harvest cells if greater than 75% of the cells appear to have died upon trypan blue viability counting.
4) Harvest cells and prepare lysates for either western blotting or immunoprecipitation. For any agent used, a time course of induction can be performed by inoculating additional dishes or flasks and harvesting at various times (i.e. 24, 48 and 72 hours) after addition of the DNA damaging agent. For the examination of apoptotic proteins, dead cells should also be collected. Always compare levels of p53 from treated cells and controls to confirm induction.

Annexin V detection protocol:Procedure for the early detection of apoptosis using annexin V, including list of available kits

2011-08-04T11:42:00.000+05:30

The appearance of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell is an early parameter of apoptosis, which can also be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and therefore can be used as a probe for detecting apoptosis.
Example protocol - Protocol for Annexin V-FITC Apoptosis Detection Kit (ab14085):

A) Incubation of cells with Annexin V-FITC

Induce apoptosis by desired method.
Collect 1-5 x 10⁵ cells by centrifugation.
Resuspend cells in 500 μl of 1X Binding Buffer.
Add 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, optional.)
Incubate at room temperature for 5 min in the dark.
Proceed to B or C below depending on method of analysis.

B) Quantification by flow cytometry

Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually FL2). For adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-FITC (A.3-5).

C) Detection by fluorescence microscopy

1. Place the cell suspension from Step A.5 on a glass slide. Cover the cells with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on a glass slide and visualize cells. The cells can also be washed and fixed in 2% formaldehyde before visualization. (Cells must be incubated with Annexin V-FITC before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane.)
2. Observe the cells under a fluorescence microscope using a dual filter set for FITC & rhodamine. Cells that have bound Annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma
membrane).
Note: The above are suggested protocols for Annexin V-FITC provided in ab14085 apoptosis detection kit. This is an example for Annexin V detection, and when using Abcam kits, the protocol provided with the kit should be used.

Annexin V detection kits:

Advances in Cytochemical Methods for Detection of Apoptosis

2011-07-21T01:12:00.001+05:30

Abstract

In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues.

he regulation of apoptosis has gained central importance in many aspects of biology, including studies of embryonic development, the pathogenesis of disease, and the response of cells to therapy (Wyllie 1992). The accurate detection of apoptosis has therefore become crucial to these studies. Beginning with the initial observations of changes in nuclear morphology that distinguished apoptosis from necrotic cell death, the field of cell death research has rapidly expanded, generating a wealth of knowledge about the genetic and biochemical pathways that control this process (reviewed in Green 2000). Several assays for detection of apoptosis were also developed during this past decade, some of which were applicable to cytochemical analysis. However, the interpretation and specificity of these assays, especially the TUNEL assay for DNA strand breaks, have been points of controversy (Allen et al. 1997). These issues have been presented in a previous article from this laboratory, in which we reviewed the available methods for apoptosis detection (Willingham 1999). The present article builds on this prior review, presents some limited new data on modifications of older methods, and reviews some newer methods for apoptosis detection.

Figure 1 summarizes a small portion of the apoptosis regulation pathways that have been recently delineated, and it is clear that this new knowledge can be the starting point for the design of more specific assays of apoptosis. Several new assays have recently appeared that depend on newly discovered changes at the molecular level. These changes are relatively more specific for the apoptotic process and offer the possibility of removing some of the controversy surrounding older detection methods. In this article, we describe our recent studies evaluating in situ nick translation (ISNT) methods using Bodipy and BUdR labeling, annexin V binding, and the detection of a caspase-specific cleavage product (cytokeratin 18) in a model system of apoptosis utilizing ricin-induced apoptosis of KB and PC3 cells. This assay system is similar to our previously presented systems in which we compared surface morphological (time-lapse phase-contrast), nuclear morphological (DAPI labeling), and ISNT (biotin-labeled) assays of apoptosis (Collins et al. 1997; Willingham 1999). Our current studies confirm the difficulties in the interpretation of the in situ end-labeling techniques (ISEL) such as ISNT (or TUNEL), and annexin V assays, but highlight the promising nature of the detection of caspase cleavage products for both cell culture and intact tissues.

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Figure 1

Diagrammatic summary of some of the biochemical steps in apoptotic pathways.

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Materials and Methods

Cells

KB is a continuous human carcinoma cell line related to HeLa cells. PC3 is a continuous human prostate carcinoma cell line. These cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI-1640 medium supplemented with 10% fetal calf serum and penicillin–streptomycin (Sigma; St Louis, MO) at 37C in a 95% air/5% CO₂ atmosphere. Cells were subcultured using 2.5% trypsin with 1 mM EDTA.

Chemicals

Annexin V labeled covalently with fluorescein isothiocyanate (FITC) was obtained from Pharmingen (San Diego, CA). Alexa 568–annexin V was obtained from Boehringer–Mannheim Biochemicals (Indianapolis, IN). DAPI (4,6-diamidino-2-phenylindole), ricin toxin, and Dulbecco's PBS were obtained from Sigma. Mouse monoclonal anti-BUdR antibody (clone B44) was obtained from Becton–Dickinson (San Jose, CA). Mouse monoclonal anti-cytokeratin 18 was obtained from Chemicon (Temecula, CA) and a mouse monoclonal antibody reactive with the caspase-cleavage product of cytokeratin 18 (M30) (Leers et al. 1999) was obtained from Boehringer–Mannheim. All secondary and tertiary labeled anti-globulin antibodies and crystalline bovine serum albumin (BSA) were from Jackson ImmunoResearch (West Grove, PA). Bodipy–dUTP was obtained from Molecular Probes (Eugene, OR) and Klenow fragment of E. coli DNA polymerase I was obtained from Sigma.

Experimental Design

Apoptosis in KB and PC3 cells was induced using ricin at 1 μg/ml as previously described (Collins et al. 1997). As shown in Figure 2, these conditions induce cell rounding in KB cells beginning within 4 hr, with all KB cells entering apoptosis in 24 hr. A subpopulation of PC3 cells entered apoptosis readily, but some PC3 cells were resistant to apoptotic entry up to the 24-hr time point (results not shown).

ISNT Labeling

ISNT was performed using a method similar to that previously presented (Collins et al. 1997). In one approach, dUTP incorporation into DNA strand breaks was assayed using Bodipy-labeled dUTP (Molecular Probes). In other experiments, we employed a modification of a BUdR labeling method (Szabo et al. 1987). Positive controls included the use of DNase treatment (1 U/μl in 1 mM Mg₂SO₄ and 1% BSA in Tris-buffered saline; 1 hr, 23C). The ISNT reactions were stopped using a 5-min incubation in 0.5 M EDTA (pH 8.0).

Cells in 35-mm dishes were induced to enter apoptosis using ricin (from 4 to 24 hr, depending on the experiment) as described above, and floating cells were recovered from the dishes, centrifuged, and resuspended in serum-free medium. The floating cells were reattached to 35-mm dishes that had been previously coated with poly-l-lysine (1 mg/ml in PBS for 5 min, washed in PBS) by centrifugation at 700 rpm (200 × g) using a swinging bucket plate rotor (Sorvall RTH-750). Both attached and adherent cells were then fixed in 3.7% formaldehyde in PBS for 10 min at 23C, followed by permeabilization with 0.2% Triton X-100 for 5 min.

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Figure 2

Detection of entry into apoptosis using video time-lapse microscopy. KB cells were induced to go into apoptosis by addition of ricin (1 μg/ml) to their medium at time 0. The flask of cells was placed on the stage of a warmed, inverted phase-contrast microscope for 24 hr and a record of cell morphology was made at a time-lapse ratio of 600:1. Entry into apoptosis was detected initially by cell rounding and blebbing. Each cell in the field was counted and the time of rounding and blebbing noted. The order of apoptotic entry is plotted on the ordinate vs the time of the initial cell blebbing on the abscissa. Two portions of the same video field were quantitated in this way and are shown as separate data points by either triangles or diamonds. The mean of the two is shown as circles. Note that 100% of the cells within the field entered apoptosis within the 24-hr time span.

The ISNT reaction mix (50 μl) using Bodipy labeling was then added to PBS-washed attached and reattached cells, and spread over the dish by overlaying the cells with a coverslip and incubating for 1 hr at 37C. The mixture consisted of water (37 μl), 10 × Klenow buffer (5 μl), 10 mM dATP, dCTP, and dGTP mix (4.5 μl), 1 mM Bodipy–dUTP (0.5 μl), and 10 U of Klenow enzyme (2 μl). After washing, the cells were postfixed in formaldehyde and mounted under a coverslip in glycerol.

For BUdR labeling, we modified the procedure of Szabo et al. (1987) as shown in Figure 3. The details of the fixation, permeabilization (0.2% Triton X-100), reaction mix, and antibody labeling are also shown in this figure. The affinity-purified secondary and tertiary antibodies were at 25 μg/ml in PBS containing 1% BSA. The volume of the reaction mixture was 1 ml or greater, allowing incubation in dishes without the use of an overlying coverslip. A key difference in this protocol that is not present in the Bodipy labeling method is the use of an acid treatment step after the ISNT reaction to render the BUdR accessible to the mouse anti-BUdR antibody. For peroxidase labeling (as in Figure 6), the secondary anti-mouse IgG was conjugated to horseradish peroxidase, rather than rhodamine, and a tertiary anti-goat IgG labeled with peroxidase was also used. This peroxidase was detected using diaminobenzidine–H₂O₂ in PBS.

Annexin V Labeling

For analysis of fluorescent annexin V binding, cells in 35-mm dishes were removed from the 37C incubations, cooled to 4C, and washed in a binding buffer containing 2.5 mM CaCl₂, 140 mM NaCl, 20 mM Tris-HCl, pH 7.5. Labeled annexin V was added to cells at 1 μg/ml in binding buffer and incubated at 4C for 1 hr. For initial experiments, the cells were then washed free of unbound annexin V and fixed in 3.7% formaldehyde at room temperature in PBS, then further incubated with formaldehyde in PBS together with 0.1 μg/ml DAPI to label nuclear DNA at 4C overnight. The attached cells were then washed in PBS and mounted under a drop of glycerol under a #1 circular 25-mm diameter coverslip in the dish. The dish was then viewed using a Zeiss Axioplan 2 upright fluorescence microscope equipped with a stage plate designed to hold 35-mm dishes.

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Figure 3

Modified BUdR-labeled ISNT protocol.

Immunocytochemistry/Immunohistochemistry

PC3 cells grown in 35-mm culture dishes were fixed using 80% acetone in water for 10 min. The cells were then incubated in 1% bovine serum albumin–PBS saline (BSA–PBS) for 10 min, then mouse anti-cytokeratin 18 (10 μg/ml; Chemicon #MAB3234) or mouse anti-caspase cleaved cytokeratin 18 (clone M30; Boehringer–Mannheim #2140322, 1:20 dilution) in BSA–PBS for 30 min at 23C. This was followed by washing in PBS, then by serial incubations in affinity-purified goat anti-mouse IgG–rhodamine, followed by rabbit anti-goat IgG–rhodamine for signal amplification (25 μg/ml; Jackson ImmunoResearch) in BSA–PBS. The dishes were then postfixed in 3.7% formaldehyde in PBS and viewed after mounting under a coverslip in glycerol. For induction of apoptosis, PC3 cells were incubated in 1 μg/ml ricin for 24 hr. To select for apoptotic cells, floating cells were collected, washed in serum-free medium, then reattached by centrifugation onto poly-l-lysine coated dishes before fixation. These cells were also subsequently stained using DAPI to visualize nuclear morphology after the final formaldehyde fixation following antibody labeling.

For immunohistochemistry using MAb M30 on paraffin sections, sections were deparaffinized routinely, treated with Antigen Unmasking Solution (Vector Labs; Burlingame, CA) while heating in a microwave for 10 min, treated with 3% hydrogen peroxide in PBS, then incubated with mouse MAb M30 (1:20 dilution in BSA–PBS) for 30 min, followed by sequential incubations with affinity-purified goat anti-mouse IgG–horseradish peroxidase, followed by rabbit anti-goat IgG–peroxidase (25 μg/ml; Jackson ImmunoResearch). The peroxidase was detected using diaminobenzidine-peroxide in PBS and counterstained with hematoxylin.

Microscopy

For time-lapse microscopy, cells in T-25 flasks were viewed under continuous incubation conditions using a Zeiss Axiovert phase-contrast microscope equipped with a warm stage and an atmospheric controller as previously described (Collins et al. 1997). Video time-lapse recordings were made at a 600:1 time lapse (one frame every 10 sec) using a Panasonic time-lapse video cassette recorder. The resulting videotape record was examined using a Macintosh 8600 computer equipped with video frame grabber software. For quantitation of apoptosis, a single frame image was printed at time 0 and individual cells were numbered. Each cell was then followed during the 24-hr video recording, and the time of its entry into apoptotic blebbing was noted.

Fluorescent samples were viewed using a Zeiss Axioplan fluorescence microscope equipped with filters for UV excitation (DAPI), blue light excitation (FITC), and green light excitation (Alexa 568 or rhodamine). The images on individual channels were captured using a SPOT cooled CCD digital camera, or a Dage 300 CCD camera. These images were either retained as grayscale images or recombined as color images with the three channels in overlay using Adobe Photoshop (4.0) software. All of the images shown in the figures represent digital images reproduced at >300 dpi.

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Results

The model system for apoptosis employed in our studies involves the induction of cell death in KB cells, a human carcinoma cell line closely related to HeLa cells. We have also used these same assays with PC3 cells, a human prostate carcinoma cell line. Apoptosis was induced in these cells through the use of ricin, the toxic lectin of the castor bean plant. Ricin binds to cells, internalizes through surface clathrin-coated pits into endosomes, and eventually translocates through intracellular membranes into the cytosol, where it irreversibly inactivates ribosomes, inhibiting protein synthesis (reviewed in Tagge et al. 1997). Whether only through this action or through other effects, ricin induces apoptosis in a wide variety of cells. In our model system, the addition of 1 μg/ml of ricin to culture medium results in the initiation of apoptotic surface blebbing in some KB cells within a few hours, and initiates apoptosis in all cells by 24 hr. An analysis of the beginning of apoptotic blebbing in two fields detected by time-lapse video phase-contrast microscopy in cultured KB cells is shown in Figure 2. This highly reproducible system allows careful comparison of the other detection methods for apoptosis at defined points in the process.

ISNT Labeling

ISNT is one method of in situ end-labeling (ISEL) for detecting strand breaks in DNA that occur during apoptosis. Although different enzymes are used in the reaction mix, ISNT and TUNEL assays are closely related. We have chosen to use ISNT rather than TUNEL because, in our experience, ISNT has usually given a stronger signal in cultured cells. Figure 4 shows the use of an ISNT assay in the KB–ricin system in which the incorporated nucleotide is directly labeled using Bodipy-conjugated dUTP. Bodipy labeling has, in our experience, produced brighter fluorescence signals in comparison to some other fluorescent markers, such as FITC. The left panels in this figure demonstrate ISNT labeling, the center panels demonstrate nuclear morphology using DAPI to label nuclear DNA, and the right panels show the phase-contrast appearance of the cells in each field. When cells undergo apoptosis in this system, cells that enter apoptosis often round up and can be washed from the culture dish. Analysis of these suspended cells provides a high frequency of apoptotic cells, while the cells attached to the plate are often not yet in apoptosis. Figure 4A shows attached cells that have been treated with DNase after fixation, rendering their nuclear DNA sufficiently fragmented to provide a positive control for the ISNT reaction. In Figure 4B, however, the otherwise untreated attached ricin-treated cells fail to show any ISNT signal.

In Figure 4C, some apoptotic cells appear rounded yet have failed to detach from the plate. Such cells show a strong ISNT signal when treated with DNase. However, in the absence of DNase treatment (Figure 4D) most of these cells fail to show an ISNT reaction. This indicates that the ISNT reaction can work in such cells but that most of these apoptotic cells are ISNT-negative at this point. To enrich for cells that have proceeded further into apoptosis, we isolated cells floating in the medium. Figire 4E shows that such cells, when reattached using poly-l-lysine-coated dishes, can be labeled with ISNT when they are treated with DNase after fixation. However, most of these cells are not labeled with ISNT in the absence of DNase treatment (arrowhead in Figure 4F). On the other hand, a few cells in the floating population show labeling with ISNT in the absence of DNase treatment (Figure 4F), and these cells have rounded morphology and segmented nuclear DNA (arrow in Figure 4F). These are the cells that would be labeled with methods such as TUNEL that detect DNA single strand breaks. It is evident from Figure 4F, however, that there are many other cells with the same morphological features (e.g., arrowheads) that fail to label with the ISNT method. Therefore, such methods appear to label only a minority of cells in a heterogeneous population at the early stages of induction of apoptosis, and confirm our prior findings that such labeling in most cells is a late event in the apoptotic process (Collins et al. 1997).

Although Bodipy labeling produces a strong signal in the ISNT reaction, the labeled substrate is rather expensive, comparable to the cost of biotin- and digoxigenin-labeled commercial substrates for ISNT and TUNEL reactions. This has often necessitated the use of the smallest possible reaction mix volumes, usually requiring the use of coverslips over cells and often resulting in damage to cells on the culture dish. We attempted to design a system that would be less expensive and allow the use of larger volumes of reaction mix. We employed the use of BUdR–triphosphate as previously described by Szabo et al. (1987). This allows a considerable decrease in cost and consequently allows the inexpensive preparation of larger volumes of reaction mix. A similar approach using terminal transferase with PAP labeling of Epon sections has been previously described (Aschoff et al. 1996). The final reaction conditions chosen are shown in Figure 3. This method utilized rhodamine fluorescence detection but could be readily adapted for other markers. The use of glycerol in the reaction mix also allows it to be frozen and re-used multiple times.

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Figure 4

ISNT of apoptotic KB cells using Bodipy labeling. KB cells were treated with ricin (1 μg/ml) for 6 hr. Then floating and adherent cells were separated and floaters were attached to new plates with poly-l-lysine, followed by fixation and permeabilization. ISNT was performed using Bodipy-labeled dUTP with (A,C,E) or without (B,D,F) treatment with DNase as a positive control for the ISNT labeling system. The dishes were post-stained using DAPI to visualize nuclear shape. Bodipy, DAPI, and phase-contrast images of the same fields are shown. As described in Results, adherent (A–D) and floating (E,F) cells showed that the majority of apoptotic cells (D,F, arrowhead) failed to show an ISNT signal at this time, although a few apoptotic floating cells were positive (F, arrow). Original magnification ×167.

Figure 5 demonstrates the results in PC3 cells, both normal and apoptotic, using this BUdR–ISNT labeling system. Interestingly, when untreated cells were examined with this system, no signal was detected in normal nuclei, as expected (Figure 5A″). However, unexpectedly, a significant signal was detected in organelles with the morphological appearance of mitochondria. We had previously detected a similar pattern using a biotin-labeled ISNT system but had assumed that this background was due to endogenous biotin in mitochondria. However, the present system does not use biotin, which suggests that this ISNT method may be labeling normal mitochondrial DNA. When these cells were treated with DNase after fixation (Figure 5B″), the nuclear DNA labeled strongly but the apparent mitochondrial signal disappeared, perhaps suggesting that the mitochondrial DNA was completely cleaved and not preserved. Apoptotic cells could be found that showed significant mitochondrial signal, but minimal nuclear signal (Figure 5C″), although some apoptotic cells were found that showed peripheral labeling of the nuclear segments (Figure 5D″). Similar to the Bodipy-labeled experiment in Figure 4, however, most apoptotic cells failed to label strongly with this ISNT method, in agreement with our previously published biotin-labeled results (Collins et al. 1997).

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Figure 5

ISNT–BUdR labeling in normal and apoptotic cells. PC3 cells were exposed to ricin, fixed, permeabilized, and reacted with the BUdR-labeled ISNT procedure described in Figure 3. Cells were either treated (B) or untreated (A,C,D) with DNase as a positive control for the labeling system. The panels shown demonstrate phase-contrast (A–D), DAPI labeling (A′–D′), and BUdR–ISNT images (A″–D″) of the same cells. (A) A normal cell with no labeling in the nucleus but labeling present in structures with a shape and distribution consistent with mitochondria. (B) A cell after DNase treatment, showing a strong ISNT signal in the nucleus (B″), and loss of the “mitochondrial” signals. (C,D) Apoptotic cells, with labeling at the edge of condensed chromatin in D″. Original magnification ×830.

We also wished to examine the use of this ISNT method using peroxidase labeling. Figure 6 demonstrates an experiment in which cells were labeled with the reaction shown in Figure 3, but subsequently labeled with peroxidase instead of rhodamine. Fixed and permeabilized normal cells showed the same “mitochondrial” pattern using HRP labeling seen with rhodamine, and cells treated with DNase as a positive control showed loss of mitochondrial labeling and gain in nuclear labeling, similar to the results using rhodamine (Figure 5). These results suggest that the BUdR–ISNT method shown is an inexpensive and accurate ISNT method useful for various labeling techniques.

Annexin V Labeling

Annexin V is a protein that binds in a calcium-dependent manner to exposed phosphatidylserine (PS). When cells undergo apoptosis, PS normally sequestered on the cytoplasmic face of the plasma membrane appears on the exterior of the cell where it can bind to labeled annexin V. Several labeled versions of annexin V are commercially available, and previous reports have described the use of this marker as an assay of apoptosis (Koopman et al. 1994; Bratton et al. 1997; Zhang et al. 1997; Gatti et al. 1998). One important issue in the use of annexin V labeling is whether the cells have proceeded through the entire apoptotic process and ultimately lysed, resulting in access to intracellular PS sites, an event that can also occur in necrotic cells. Another issue related to annexin V binding is whether all apoptotic cells externalize PS or whether only a sub-population of apoptotic cells do so.

We examined annexin V binding during apoptosis with fluorescently-labeled derivatives using either FITC or Alexa 568 as marker in the KB–ricin model system. Figure 7 shows KB cells treated with ricin for 6 hr, then labeled with FITC–annexin V. Apoptotic cells were easily identified on the basis of their rounded, blebbing morphology and the segmentation of their nuclei detected using DAPI. Some of these cells subsequently incubated with annexin V at 4C showed surface binding of FITC-labeled annexin V, but a significant number failed to show surface labeling (Figure 7A′). We quantitated this event at various times of ricin treatment, as shown in Figure 8. After a lag of 4 hr, cells began to show rounded morphology and altered nuclear shape, and a fraction of these same cells labeled with annexin V. Even at 22 hr after addition of ricin, however, when almost all cells have entered apoptosis, only about one third of the cells labeled with annexin V, even though each of these unlabeled cells showed the other surface and the nuclear morphological features of apotosis.

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Figure 6

ISNT–BUdR in normal PC3 cells using peroxidase labeling. Untreated PC3 cells were fixed and incubated as shown in Figure 5, except that the ISNT–BUdR reaction was detected with peroxidase-conjugated antibodies rather than rhodamine. The same apparent mitochondrial signal was detected in normal cells (A,A′) as demonstrated by both phase-contrast (A,B) and brightfield images (A′,B′). DNase treatment resulted in loss of the mitochondrial signal and gain of a nuclear signal (B,B′). Original magnification ×200.

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Figure 7

Detection of annexin V binding to the surface of apoptotic cells. KB cells were incubated with ricin (1 μg/ml) at 37C. At 6 hr, dishes of cells were removed from the incubator, cooled to 4C, washed, and incubated with FITC-labeled annexin V (1 μg/ml) for 1 hr. The cells were then washed in PBS and fixed in formaldehyde, together with DAPI, in PBS, followed by viewing using fluorescence microscopy. The same field of cells is shown in all of the panels. (A) Phase-contrast image; (A′) the image on the fluorescein channel; (A″) the image on the DAPI channel. Note that some blebbing cells show strong annexin V labeling of their surfaces (arrow) as well as nuclear segmentation (A″). However, some cells (arrowheads) show blebbing (A) and nuclear segmentation (A″) but no annexin V labeling (A′). Original magnification ×175.

One possible explanation for only a minority of apoptotic cells labeling with annexin V at these times was that our assay might have been performed at too early a time, and that cells might show progressive annexin V labeling at later times. To test this idea, we performed a double-label annexin V experiment in which KB cells were labeled with Alexa 568-conjugated annexin V at 6 hr after ricin addition, washed, and allowed to incubate at 37C for an additional 3 hr, and then labeled with FITC-conjugated annexin V. Figure 9 shows results from this experiment, in which some apoptotic cells failed to label at either 6 or 9 hr (Figure 9A, arrowheads), others labeled at both 6 and 9 hr (Figures 9A and 9B, arrow), and still others labeled only at 9 hr (Figure 9B, arrowhead). This might suggest that some apoptotic cells progress in their externalization of PS as they proceed through apoptosis.

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Figure 8

Quantitation of morphological changes and annexin V binding in apoptotic cells. Cells in dishes from the experiment shown in Figure 7 were also examined individually for apoptotic morphology, nuclear segmentation, and annexin V binding at various times after ricin addition. At least 50 cells were examined and counted in each group. The percent of the total number of cells in the dish that showed these changes are plotted on the ordinate, with individual incubation times for each dish shown on the abscissa. Note that apoptotic morphological changes appear at 4 hr and, at that point, only about one third of those cells also show annexin V binding. This trend is also seen at a later time point (22 hr), at which most cells have begun apoptosis. Even then, only about one third of cells show annexin V binding. Note also, however, that the percentage of cells showing annexin V binding appears to increase between 4 and 6 hr, when the total percentage of apoptotic cells has increased only slightly. This suggests that apoptotic cells negative for annexin V binding might progress at a later time to show annexin V binding. Error bars represent 1 SD for each of the individual groups.

If cells failed to label at 6 hr, it could also be due to the fact that they had not yet begun apoptosis. Therefore, we repeated this experiment using mechanical shaking selection to enrich for floating cells, in which almost all of the harvested cells had entered apoptosis on the basis of their surface morphology (results not shown). When we labeled these cells at 6 hr with Alexa–annexin V, washed, then labeled later at 9 hr with FITC–annexin V, we saw the results shown in Figure 10. Figures 10A″ and 10B″ are composite overlays of the images on individual channels shown in Figures 10A′-A″ and 10B′-B″. Some cells showed apoptotic surface and nuclear morphology and did not label at either time with annexin V (results not shown). Other cells labeled at 9 hr but not at 6 hr (Figure 10B, arrow). In addition, some cells labeled at both 6 hr and 9 hr (Figure 10A). Interestingly, cells that had labeled at 6 hr showed internalization of the Alexa-labeled annexin V (Figure 10A″), indicating that apoptotic cells continue to endocytose material from their surfaces even though they have already begun to show other morphological changes typical of apoptosis. In other words, even though the cells have begun the process of dying, they still demonstrate at least one activity typical of living cells. Figure 10 also shows a cell (arrowhead) that demonstrates an interpretive problem with the use of annexin V, in that this cell has proceeded through the final lysis stage of apoptosis, allowing intense labeling of its internal PS directly. Such a result would also be seen with necrotic cells.

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Figure 9

Double-label detection of annexin V binding at different times in the same cells. As described in Materials and Methods and in Results, cells were induced with ricin, then incubated with annexin V labeled with Alexa 568 at +6 hr, then incubated further at 37C until + 9 hr, when they were incubated with FITC-labeled annexin V, followed by fixation. Phase-contrast images are shown in (A,B), DAPI channel images in (A′,B′), Alexa channel images in (A″), and fluorescein channel images in (A′″). Note the central apoptotic cell in A (arrow) that shows altered nuclear morphology in A′, internalized Alexa–annexin V in A″, and also surface FITC–annexin V in A′″. Other apoptotic cells (arrowheads) show altered nuclear morphology (A′) but failed to label with annexin V at either 6 (A′) or 9 (A′″) hr. On the other hand, note the cell in B (arrowhead) which shows nuclear segmentation in B′, FITC–annexin V labeling in B′″, but no Alexa–annexin V labeling in B″, suggesting that it did not label with annexin V at +6 hr. An apoptotic cell labeled at both times is shown by the arrow in B–B′″. Original magnification ×265.

Detection of Caspase-generated Cleavage Products: Cytokeratin 18

Because caspase activity is likely to be the most specific indicator of the apoptotic process, assays of caspase activity through the detection of specific cleavage products in target proteins should represent a promising approach for measuring apoptosis. Such an approach could be applicable to fixed archival tissue blocks. We tested a recently commercially available antibody to the caspase-generated cleavage products of cytokeratin 18 (antibody M30) in PC3 cells, which can be shown to contain cytokeratin 18. This antibody (Leers et al. 1999) is similar in concept to other antibodies to cleaved keratins that have been previously reported (Caulin et al. 1997). Figure 11 shows the presence of cytokeratin 18 in untreated PC3 cells (Figure 11A′) using an antibody to intact cytokeratin. Figure 11 shows non-apoptotic cells from an experiment in which ricin was added to induce apoptosis in this same cell type. No reaction of MAb M30 was observed in such non-apoptotic cells. On the other hand, most cells showing apoptotic morphology (Figures 11C-11E) showed reaction with M30 in their cytoplasm, indicating the presence of caspase-cleavage products of cytokeratin 18. These results suggest that such an assay is potentially very useful for detecting apoptotic cells in cell culture. To confirm that this antibody could also be applied to fixed tissue sections, we examined M30 reactions detected by peroxidase labeling in an archival sample of human colon carcinoma. Figure 12 shows the positive reaction of this antibody selectively with cells with apoptotic morphology, suggesting that such an antibody is also useful in archival embedded tissues.

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Discussion

Apoptosis is a central element in the pathogenesis of many disease processes and in the response to systemic therapies in neoplastic cells. Specific detection of apoptosis in tissue cells is therefore an important cytochemical technique for biomedical studies. The methods developed over the past decade for the detection of apoptosis have often been derived from empirical observations of the properties of dying cells. Recently, however, the molecular and biochemical mechanisms of apoptotic death have been further defined, leading to the prospect of assay methods based on highly specific chemical changes (Green 2000). Of the assays previously employed for apoptosis detection, those that are applicable to intact preserved tissues would have the broadest utility. One approach, the detection of DNA fragments through TUNEL or ISNT techniques, has been used extensively. Although useful, there are issues of specificity and interpretation of such techniques that have inspired a search for more highly specific assays.

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Figure 10

Double-label detection of annexin V binding in disattached apoptotic cells. To confirm that the cells examined in Figure 9 were actually in apoptosis at +6 hr, the same experiment was repeated, but with selection (by shaking) of rounded disattached cells at the 6-hr time point. The cells were then further incubated until +9 hr and incubated with FITC–annexin V. In A–A′″ note the rounded morphology by phase contrast in A, the nuclear segmentation on the DAPI channel in A′, the previously internalized Alexa-568-labeled annexin V in A″, and the surface labeling by FITC–annexin V at +9 hr in A′″. On the other hand, note the cell labeled with an arrow in B. This cell shows no Alexa 568 labeling (B″), but strong FITC–annexin V labeling (B′″), suggesting that this cell was apoptotic at +6 hr (based on selection by shaking) but that it had not yet externalized PS to its surface. The cell shown by the arrowhead has progressed further in apoptosis to the lysis stage and labels internally with FITC–annexin V (B′″). Also note that the intact cells in A″ and B″ show internalized Alexa–annexin V which was originally present on the cell surface at +6 hr. This demonstrates that, even though the cells had become apoptotic before +6 hr, they continued endocytosis at least until +9 hr. A″″ and B″″ are color overlays of the separate channels shown as grayscale images in A′, B′, A″, B″, and A′″, B′″. Original magnification ×367.

In this study, we have used a defined cell culture system, ricin-induced apoptosis in KB and PC3 cells, in which apoptosis can be induced in a highly synchronous and reproducible manner. The characteristics of this process are easily detectable at both the morphological and the biochemical level. We have applied some of the more promising recent cytochemical assays to this system. Because TUNEL and ISNT assays are perhaps the most commonly used cytochemical methods in this field, we have attempted to develop a less expensive alternative ISNT method to allow the use of larger reaction volumes for these assays. Through modification of an existing BUdR labeling method, we tested this method in the ricin-induced apoptosis system and found results similar to those previously reported (Collins et al. 1997), in which the ISNT reactions were seen as a late event in the apoptotic process in a minority of cells.

We also examined another commonly used cytochemical assay of apoptosis, the labeling of externalized PS by annexin V. Although this assay detects apoptosis at an earlier stage than ISNT, only a minority of apoptotic cells actually labeled with annexin V before the final lysis of cells at the end of the apoptotic process. This is in agreement with other studies published previously using a different apoptosis system (Gatti et al. 1998). The annexin V experiments did demonstrate, however, that cells appear to progress in their externalization of PS with time, and that cells already involved in the apoptotic process still demonstrate endocytic activity for many hours.

Finally, we examined one newly available antibody that detects the specific cleavage fragments of a cytoplasmic target of caspases, cytokeratin 18 (Leers et al. 1999). Not only did this antibody detect apoptotic cells with high specificity in cell culture but it also showed reaction in archival tissue sections. Cytokeratin 18 is expressed only in certain cell types, however, and this antibody is not broadly applicable to all cell types. The use of antibodies specific for more generally distributed cleaved substrates of caspases, such as the cleaved form of caspase 3, would have more general applicability.

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Figure 11

Detection of caspasecleaved cytokeratin 18 in cultured cells. PC3 cells were initially demonstrated to contain intact cytokeratin 18 using an antibody reactive with the intact protein (A′) using immunofluorescence. PC3 cells were then treated with ricin and examined using an antibody that reacts selectively with caspase-cleaved cytokeratin 18 (M30). Non-apoptotic cells in this culture failed to react with this antibody (B″) but apoptotic cells showed a bright cytoplasmic speckled pattern (C″–E″). [A–E, phase-contrast; A′, anti-cytokeratin 18 (intact); B–E′, DAPI; B″–E′, anti-cleaved cytokeratin 18 (antibody M30)]. Original magnifications: A ×250; B–E ×600.

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Figure 12

(A-D) Detection of caspase-cleaved cytokeratin 18 in paraffin sections. Sections of formalin-fixed, paraffin-embedded human colon adenocarcinoma were reacted with anti-cleaved cytokeratin 18 (M30) and labeled using peroxidase. These images show examples (arrows) of cells with apoptotic morphology that label strongly using this antibody. Original magnification ×320.

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Acknowledgments

We wish to thank Drs Timothy Kute and Arthur Frankel for helpful discussions. These results include data generated while JMW was the recipient of a summer student scholar research award from the South Carolina Governor's School.

Received November 17, 2000.
Accepted March 7, 2001.

1. ¹Department of Pathology, Wake Forest University School of Medicine, Winston–Salem, North Carolina
1. Correspondence to: Mark C. Willingham, Dept. of Pathology, Wake Forest Univ. School of Medicine, Gray Bldg., Room 2106, Medical Center Blvd., Winston–Salem, NC 27157. E-mail: mwilling@wfubmc.edu

INDO-1 LOADING AND SAMPLE STAINING PROCEDURE FOR SIMULTANEOUS MEASUREMENT OF INTRACELLULAR CA2+ AND CELL SURFACE ANTIGEN EXPRESSION

2011-07-21T01:05:00.000+05:30

MATERIALS:

1. 50 mg vial Indo-1 (Cat # I-1203, Molecular Probes, OR)
2. DMSO (Sigma, St. Louis, MO)
3. RPMI 1640
4. Monoclonal antibodies (mAb), conjugated to suitable fluorochromes
5. Ionomycin (Calbiochem, San Diego, CA)
6. 37°C water bath, centrifuge, vortexer.
7. Agonists to test Ca2+ flux, e.g. anti-CD3, anti-IgG, ConA.
8. Serum (for RPMI with 2% serum, if cells require serum).

METHOD:

1. Incubate cells (£2x107/ml) in RPMI with 1-5 mM Indo-1 (acetoxymethyl ester) at 37°C for 40 min for loading.

2. Incubate aliquots of Indo-1 loaded cells with saturating concentrations of e.g., FITC, PE, PerCP, or Tricolor-conjugated antibodies for 20 min. Incubate at 20° to 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC. Note: mAbs must be azide free. Note: set-up single color stained cells for setting appropriate fluorescence compensation on the instrument.

3. Wash cells twice in RPMI and suspend them at the desired concentration (usually 2x106/ml). Higher cell concentrations (4x106/ml) are required when the cells of interest represent less than 10% of the total population. Cells can be kept at 20° to 25oC unless the antigen is subject to capping, otherwise use 4° to 8oC.

4. Samples should be analyzed shortly after the cells were prepared.

5. Ionomycin (1-3mM final conc.) is used as a positive control for Indo-1 loading and maximum Ca2+ flux.

PREPARATION OF INDO-1:

1. Add 150 ml of DMSO to a 50 mg vial of Indo-1, cover with aluminum foil to protect from light.
2. Vortex well, then warm to 37°C for 5 min.
3. Transfer 150 ml of Indo-1 from vial to 4.85 mls of RPMI (=10mM). Wash out vial very well. If not the entire amount of Indo-1 dissolved in DMSO is used, the remainder can be stored dessicated at –20oC for less than 6 months.
4. Cover the tube of 5 ml of 10 mM Indo-1 with foil.
5. Aliquot the appropriate amount of 10mM Indo-1 to the cell suspension (final conc.=1-5 mM). The optimal concentration is dependent on the cell type.
6. Store excess RPMI-diluted 10 mM Indo-1 at 4°C. In our laboratory, the 10 mM Indo-1 solution has been tested for stability up to 24 hrs.

PREPARATION OF IONOMYCIN:

1. Dissolve 1 mg of ionomycin in 1 ml DMSO.
2. Aliquot 13.5 ml of ionomycin solution into vials for later use and store at -20°C for less than one year.
3. Dilute one 13.5ml vial of ionomycin with RPMI to a volume of 3 mls (=6 mM).
4. Cover the 3 mls of 6 mM working stock with foil to protect from light.
5. 150 ml of working stock ionomycin is added to 300 ml of Indo-1 loaded cell suspension.

Special Note:
Indo-1 requires UV excitation. Make sure that you have an instrument with either an argon laser tuned to UV or a helium-cadmium laser available. Because experiments involving the measurement of calcium flux are performed directly on the flow cytometer, pre experiment consultation is strongly recommended.

Further Reading:
June CH, Rabinovitch PS. Intracellular ionized calcium. Methods Cell Biol. 1994;41:149-74.

June CH, Abe R, Rabinovitch PS. Measurement of intracellular calcium ions by flow cytometry. In: Current Protocols in Cytometry, Vol 2, Robinson JP, Darzynkiewicz Z, Hyun W, Orfao A, Rabinovitch P, eds., John Wiley & Sons, 1997, pp. 9.8.1 – 9.8.19.

Simultaneous measurement of three-color immunofluorescence and DNA content

2011-07-21T01:02:00.000+05:30

Materials
Phosphate buffered saline (1 X PBS without Ca++ and Mg++)
Newborn calf serum (NCS)
Sodium azide (NaAz)
Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe
Dimethylsulfoxide (DMSO)
Saponin (Sigma)
7-amino-actinomycin D (7-AAD) stock solution, see recipe
Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe
Method
1. Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 µl of PBS supplemented with 2% NCS and 0.1% NaAz and mix well.
2. For staining of cell surface antigen expression add appropriate amounts of biotinylated, phycoerythrin (PE)-labelled and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20oC - 25oC.
3. Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min.
4. Remove the supernatant and add 100 µl of PBSAz containing 2 µg of streptavidin Alexa Fluor488R and follow by incubation for 20 min at 4oC. Wash cells once with 2 ml of PBSAz by centrifugation at 250 x g for 5 min at 4oC.
5. Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin and 10 µg/ml of 7-AAD followed by incubation for 20 min at 20oC - 25oC protected from light.
6. Then, spin cells down by centrifugation at 250 x g for 5 min. Resuspend the cell pellet in 0.5 ml of NASS containing 0.02% of saponin and 10 µg/ml of AD and place the mixture on ice protected from light for at least 10 min before acquisition on the flow cytometer.

Note: it is possible to replace cell surface staining with Alexa Fluor 488 which is not pH sensitive with staining using mAbs directly labelled with FITC and omit the second staining step with streptavidin; however, to restore the FITC fluorescence that is markedly diminished in NASS at pH 4.8, it is necessary to resuspend cells after the spin in step 6 in 1 X PBS at pH 7.2 containing 0.02% of saponin and 10 µg/ml of AD and place them on ice for at least 10 min before acquisition on the flow cytometer.

Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 µl of DMSO, then add 950 µl of 1 X PBS; keep at 4oC protected from light.

Nucleic acid staining solution (NASS, pH 4.8): 0.15 M NaCl in 0.1 M phosphate-citrate buffer containing 5 mM sodium EDTA and 0.5% BSA fraction V

Dissolve 2 tablets of phosphate-citrate buffer in 100 ml of distilled H20 to make a 0.1 M solution.

Add 0.18 g of disodium EDTA to a final concentration of 5 mM.

Add 0.9 g of NaCl to a final concentration of 0.15 M.

Add 0.5 g of BSA to a final concentration of 0.5%.

Keep at 4oC.

Actinomycin D (AD) stock solution (1mg/ml): dissolve 1 mg of AD powder first in 50 µl of DMSO, then add 950 µl of 1 X PBS, keep at 4oC protected from light.

Reference:

Schmid I, Cole SW, Zack JA, Giorgi JV. Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry. J Immunol Meth. 235:121-131, 2000.

Simultaneous measurement of dual-color immunofluorescence and DNA/RNA content

2011-07-21T00:59:00.002+05:30

Materials

Phosphate buffered saline (1 X PBS without Ca++ and Mg++)

Newborn calf serum (NCS)

Sodium azide (NaAz)

Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe

Dimethylsulfoxide (DMSO)

Saponin (Sigma)

7-amino-actinomycin D (7-AAD) stock solution, see recipe

Pyronin Y(G) (PY) (Polysciences)

Actinomycin D (C1) (AD, Roche Molecular Biosystems) stock solution, see recipe

Method

Place 1 x 106 PBS-washed cells into a 12 x 75 mm tube and add 100 µl of PBS supplemented with 2% NCS and 0.1% NaAz and mix well.

For staining of cell surface antigen expression add appropriate amounts of biotinylated and allophycocyanin (APC)-labelled monoclonal antibodies (mAb) or of corresponding labelled isotypic control antibody and incubate the samples while protected from light for 15 min at 20ºC - 25ºC.

Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min.

Remove the supernatant and add 100 µl of PBSAz containing 2 µg of streptavidin Alexa Fluor488R and follow by incubation for 20 min at 4ºC. Wash cells once with 2 ml of PBSAz by centrifugation at 250 x g for 5 min at 4oC.

Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin and 10 µg/ml of 7-AAD followed by incubation for 20 min at 20ºC - 25ºC protected from light.

Then, add 1 ml of 1 X PBS and spin cells down by centrifugation at 250 x g for 5 min. Resuspend the cell pellet in 0.5 ml of NASS containing 0.02% of saponin and 10 µg/ml of AD and place the mixture on ice protected from light for 5 min.

Add 0.5 µl of a 1mg/ml stock solution of PY in distilled water. Vortex immediately and keep on ice protected from light for at least 10 min before sample acquisition on the flow cytometer.

Note: it is possible to keep cells for a maximum of three days protected from light at 4ºC in the staining solution without adverse effects.

Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 µl of DMSO, then add 950 µl of 1 X PBS; keep at 4ºC protected from light.

Nucleic acid staining solution (NASS, pH 4.8): 0.15 M NaCl in 0.1 M phosphate-citrate buffer containing 5 mM sodium EDTA and 0.5% BSA fraction V Dissolve 2 tablets of phosphate-citrate buffer in 100 ml of distilled H20 to make a 0.1 M solution.

Add 0.18 g of disodium EDTA to a final concentration of 5 mM.

Add 0.9 g of NaCl to a final concentration of 0.15 M.

Add 0.5 g of BSA to a final concentration of 0.5%. Keep at 4ºC.

Actinomycin D (AD) stock solution (1mg/ml): dissolve 1 mg of AD powder first in 50 µl of DMSO, then add 950 µl of 1 X PBS, keep at 4ºC protected from light.

Reference: Schmid I, Cole SW, Korin YD, Zack JA, Giorgi JV. Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence. Cytometry 39:108-116, 2000.

Simultaneous measurement of cell surface immunofluorescence, viability, and DNA content

2011-07-21T00:58:00.002+05:30

Materials

Phosphate buffered saline (1 X PBS without Ca++ and Mg++)Newborn calf serum (NCS)

Sodium azide (NaAz)

Nucleic acid staining solution (NASS, phosphate-citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), all from Sigma), see recipe

Ribonuclease A (RNAse)

Dimethylsulfoxide (DMSO)

Saponin (Sigma)

7-amino-actinomycin D (7-AAD) stock solution, see recipe

TO-PRO-3 iodide (TP3) (Molecular Probes), note that TP3 requires a flow cytometer capable of red excitation, e.g., 633 nm, 635 nm

Actinomycin D (C₁) (AD, Roche Molecular Biosystems) stock solution, see recipeµ

Method

Place 1 x 10⁶ PBS-washed cells into a 12 x 75 mm tube and add 250 µl of PBS supplemented with 2% NCS and 0.1% NaAz and containing 4 µg/ml of 7-AAD and mix well.
For staining of cell surface antigen expression add appropriate amounts of FITC-labeled and PE-labeled monoclonal antibodies (mAb) or of FITC and PE isotypic control antibody and incubate the samples while protected from light for 15 min at 20^oC - 25^oC.
Wash cells once with 2 ml of 1 X PBS by centrifugation at 250 x g for 5 min. Remove the supernatant immediately and completely and add 2 ml of 1 X PBS containing 4 µg/ml of AD. Vortex the mixture immediately, spin cells down for at least 5 min at 250 x g, and remove the supernatant completely.
Resuspend cells in 0.5 ml of NASS containing 0.02% of saponin, 4 µg/ml of AD, 0.5 µM of TP3, and 200 µg/ml of RNAse followed by incubation for 30 min at 20^oC - 25^oC.
If samples were cell surface stained only with PE-labelled mAbs, they are acquired on the flow cytometer in their staining solution after the incubation.
If samples were cell surface labelled with FITC-conjugated mAbs alone or with FITC and PE-labelled mAbs, spin cells down after DNA staining by centrifugation at 250 x g for 5 min; then, resuspend the cell pellet in 0.5 ml of 1 X PBS at pH 7.2 containing 0.02% of saponin, 0.5 µM of TP3, and 4 µg/ml of AD to restore the FITC fluorescence that is markedly diminished at pH 4.8. Then, acquire samples on the flow cytometer in their staining solution.

Preparation of solutions:

7-AAD stock solution (1mg/ml): dissolve 1 mg of 7-AAD powder first in 50 ml of DMSO, then add 950 ml of 1 X PBS; keep at 4°C protected from light.
Nucleic acid staining solution (NASS, pH 4.8): 0.15 M NaCl in 0.1 M phosphate-citrate buffer containing 5 mM sodium EDTA and 0.5% BSA fraction V.
Dissolve 2 tablets of phosphate-citrate buffer in 100 ml of distilled H₂O to make a 0.1 M solution.
Add 0.18 g of disodium EDTA to a final concentration of 5 mM.
Add 0.9 g of NaCl to a final concentration of 0.15 M.
Add 0.5 g of BSA to a final concentration of 0.5%.
Keep at 4°C.

Actinomycin D (AD) stock solution (1mg/ml): dissolve 1 mg of AD powder first in 50 µl of DMSO, then add 950 µl of 1 X PBS, keep at 4^oC protected from light.

Reference:

Schmid I, Hausner MA, Cole SW, Uittenbogaart CH, Giorgi JV, Jamieson BD. Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. J Immunol Meth. 247:175-186, 2001.

Measurement of Green Fluorescent Protein Expression and DNA Content in Unfixed Cells

2011-07-21T00:57:00.002+05:30

Reagents

Cells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as control.

Hoechst 33342 stock solution (1mg/ml) (see recipe)

12 X 75 mm culture tubes
Vortex mixer

Waterbath at 37oC

Method

1. Count cells.

2. Place approximately 106 cells into a 12 x 15 mm test tube and spin them down by centrifugation for 5 min at 300 x g.

3. Remove supernatant by aspiration or rapid decanting and add 500 ml of the medium that was used for growing the cells to be studied pre-warmed to 37oC to the cell pellet. Mix gently. Add 5 ml of Hoechst 33342 stock solution and mix again. Incubate at 37oC for 45 min.

The optimal Hoechst dye concentration and staining time for different cell types vary as dye up-take depends on cellular metabolic rates; thus, both have to be determined empirically. In general, dye concentrations between 1mg/ml and 10 mg/ml and incubation times between 20 min and 90 min will produce DNA histograms with acceptable coefficients of variation. Because Hoechst DNA staining is performed on unfixed cells, it is possible to use other non-vital DNA dyes, e.g., PI, 7-aminoactinomycin D (7-AAD), for concurrent dead cell discrimination.

Preparation of Hoechst 33342 stock solution:

Dissolve 1 mg of Hoechst 33342 powder (Molecular Probes, Eugene, OR) in 1 ml of distilled water. Store at 2-8oC protected from light for up to 1 month.

Reference

Schmid I. and Sakamoto KM. Analysis of DNA content and green fluorescent protein expression. In: Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z, Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John Wiley & Sons, 2001, pp. 7.16.1-7.16.10.

Measurement of GFP Expression and DNA Content in Permeabilized Cells

2011-07-21T00:55:00.000+05:30

Reagents

Cells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as a control.

1 X PBS
2% Buffered formaldehyde solution (see recipe)
70% Ethanol
Propidium iodide stock solution (1mg/ml in PBS)
DNAse-free ribonuclease A
2 X 75 mm culture tubes
Vortex mixer
Waterbath at 37oC

Method

Fix cells with formaldehyde

1. Count cells.
2. Place approximately 106 cells into a 12 x 15 mm test tube and wash them once with PBS by centrifugation for 5 min at 300 x g at 2-8oC.
3. Remove supernatant by aspiration or rapid decanting and add 500 ml of cold PBS to the cell pellet. Mix gently. Add 500 ml of cold, buffered 2% formaldehyde solution and mix again. Incubate at 2-8oC for 1h.

Permeabilize cells with ethanol

4. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, remove supernatant by aspiration or rapid decanting, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at – 20 oC drop-wise to the cell pellet with the tube sitting on a vortex. Incubate cell suspension overnight at 2-8oC.

5. Spin cells down by centrifugation for 5 min at 300 x g at 2-8oC, remove supernatant by aspiration or rapid decanting and add 1 ml of a solution containing 40µg/ml of PI and 100 µg/ml of ribonuclease A. Incubate cell suspension at 37oC in the dark for 30 min. If needed, filter samples through a nylon mesh to remove clumps before acquisition on the flow cytometer.

Preparation of Buffered 2% Formaldehyde Solution

Add 2g paraformaldehyde (Polysciences, Inc.) to 100 ml PBS. Heat the solution to 70oC in a fume hood until the paraformaldehyde goes into solution (approximately 1 h ). Cool to room temperature, check pH and adjust to 7.2 with 0.1M NaOH or 0.1M HCl. Store at 2-8oC protected from light. The solution is stable for at least 1 month. Check pH periodically. Do not heat the solution above 70oC. For best results, use only very pure preparations of paraformaldehyde (i.e., electron microscopy grade from Polysciences).

References

Chu, YW, Wang R., Schmid I, Sakamoto KM. Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. Cytometry 36:333-339, 1999.

Schmid I. and Sakamoto KM. Analysis of DNA content and green fluorescent protein expression. In: Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z, Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John Wiley & Sons, 2001, pp. 7.16.1-7.16.10.

Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements

2011-07-21T00:52:00.000+05:30

ACCURATE MEASUREMENTS OF FLUORESCENCE INTENSITY SHIFTS CAN BE MADE BY CALIBRATING THE CYTOMETER WITH CHICKEN RED BLOOD CELLS¹
BACKGROUND

Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels.
MATERIALS
1. 10% Bleach solution
2. Double distilled water
3. Unstained cells of the type you are going to analyze in your experiment (negative control sample)
4. Cells stained with monoclonal antibodies of interest (positive control sample)
5. Glutaraldehyde-fixed CRBCs (Biosure, Cat#1004, Riese Enterprise. San Jose, CA)
6. 1x PBS + 2% newborn calf serum + 0.1% Sodium azide (PBSAz)
METHODS
ESTABLISH PMT VOLTAGE SETTINGS FOR YOUR SAMPLES

1. Clean the flow cytometer fluid lines by running 10% bleach for 5 minutes, followed by distilled water for 5 minutes.

2. Launch the CellQuest "Three Color Acquisition Screen" template and set up the software for data acquisition.

3. Put FSC and SSC detectors in LIN mode. Put fluorescence parameter(s) in LOG mode.

4. Set all compensation values to zero.

5. Place the negative-control sample onto the flow cytometer and start acquisition in setup mode.

6. Adjust the FSC and SSC settings so that the cell cluster appears on scale.

7. Position your negative control cluster in appropriate location by adjusting PMT voltages. This is generally within the first decade of the log scale with the cluster fully visible.

8. Put on the positive control sample and make sure the positive signal is not off scale. If so, decrease PMT settings.

9. Make note of final PMT settings for FL-1, FL-2 and FL-3.

DETERMINE CRBC TARGET CHANNELS

10. Put two drops of concentrated CRBC resuspension into a 12x7.5 millimeter tube. Add 1 ml PBSAz and mix well.

11. Open the CellQuest template "CRBC SETUP"² located on the desktop and set up for acquisition.

12. Acquire the CRBCs in setup mode.

13. Adjust the FSC and SSC settings to bring the CRBCs on scale and set all compensation values to zero. Use the PMT voltage settings from #9 above.

14. Position the histogram markers on each peak to calculate the mean channel values for each parameter.

15. Record the mean channel values for each parameter. These are your target channels. Save these instrument settings in an appropriately labeled folder on you disk. Now that your target channels have been determined, you need to calibrate the machine to your own target channels prior to each fluorescence intensity experiment. Note that different lot numbers of CRBCs will yield different target channels; therefore, these need to be re-determined for each lot of CRBCs.

CALIBRATION FOR FLUORESCENCE INTENSITY MEASUREMENTS

16. Clean the machine with bleach and water as described in 1.

17. Prepare the CRBC solution as described in 2.

18. Launch CRBC SETUP template and set up for acquisition. Use instrument settings from #9 (same as #15). Compensation settings must all be zero.

19. Make necessary adjustments to PMT voltages so that the histogram mean channels for each parameter are the same as your target channels (#15). Restart acquisition after each PMT adjustment.

20. Record PMT voltages.

FLUORESCENCE INTENSITY MEASUREMENTS

21. Launch "Three Color Acquisition Screen" and set up for acquisition.

22. Adjust FSC and SSC settings to bring cells on scale.

23. Set PMTs to same voltage as needed to calibrate with CRBCs (#20).

24. Set fluorescence compensation as necessary.

25. Acquire data. Mean channel intensity shifts will be due to receptor expression level changes and not due to instrument variability.

¹This protocol will allow for RFI comparisons only in experiments that utilize the same cells and the same treatments.

²The CRBC Setup document consists of one FSC versus SSC dot plot and one non-gated histogram (with histogram statistics) for each fluorescence parameter. A marker is placed on each histogram to generate accurate mean channel statistics for each parameter. The histograms should have channel values displayed (Plots menu»Log Data Units»Channel Values).

MEASURING APOPTOSIS AND NECROSIS BY DUAL LASER FLOW CYTOMETRY

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MATERIALS:

1. 1 X PBS (PBS-BSA, without sodium azide + 2% bovine serum albumin)
2. Human AB serum, heat inactivated (HAB, e.g., Irvine Scientific, CA)

3. Hoechst 33342 (HO342, Molecular Probes, OR)

4. Propidium iodide (PI, e.g., Calbiochem, CA)

5. 7-Amino-actinomycin D (7-AAD, e.g., Calbiochem, CA)

6. 37°C waterbath, centrifuge

7. Bucket with ice

Preparation of stock solutions of HO342, PI, and 7-AAD

HO342

Dissolve HO342 powder at a concentration of 1mg/ml in distilled H2O. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 months.

PI

Dissolve PI powder at a concentration of 1mg/ml in 1 X PBS. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 months.

7-AAD

Dissolve 7-AAD powder (1mg) first in 50 microliters of absolute methanol, then add 950 microliters of 1 X PBS. Final concentration is 1mg/ml. Keep solution at 4°C protected from light. Solution can be stored for at least up to 6 months.

METHOD:

Principle:

Apoptotic cells, due to a change in membrane permeability, show an increased up-take of the vital dye HO342 compared to live cells. PI or 7-AAD is added to discriminate late apoptotic or necrotic cells which have lost membrane integrity from early apoptotic cells which still have intact membranes by dye exclusion.

References:

Ormerod MG, Collins MKL, Rodriguez-Tarduchy G, Robertson D: Apoptosis in Interleukin-3 dependent haemopoietic cells: quantification by two flow cytometric methods. J Immunol Meth 153:57-65, 1992.

Schmid I, Uittenbogaart CU, Giorgi JV: A sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15:12-20, 1994.

Staining for detection of apoptosis

1 x 106 PBS-BSA-washed cells from a single cell suspension are pelleted in a 12 X 75mm culture tube. The pellet is resuspended in 1ml of PBS-BSA pre-warmed to 37°C and the suspension is mixed gently. Then, 1 microliter of HO342 stock solution is added and the mixture is immediately vortexed briefly. The suspension is incubated for exactly 7 min at 37°C, then placed on ice, and 1 microliter of PI stock solution or 1 microliter of 7-AAD stock solution is added. After approximately 10 min the samples can be run on the flow cytometer. They should be kept cold during acquisition and should be run within 1 hour after the addition of the DNA dyes.

This staining procedure allows the correlation of apoptosis with single or dual-color phenotype. Surface antigen staining is done according to standard methods before staining for apoptosis. Then, the apoptosis assay is performed as described above.

Direct Staining Procedure

1. Resuspend cell pellet first in 50 microliters of HAB for approximately 1 min, then add 50 microliters of PBS-BSA and the appropriate amount of fluorochrome-conjugated monoclonal antibody (mAb).

2. Vortex briefly and incubate for 15 min at 4°C in the dark.

3. Wash once with 2 mls of PBS-BSA by centrifugation at 250g for 5 min.

4. Resuspend samples in 1 ml of PBS-BSA pre-warmed to 37°C.

5. Proceed with staining for apoptosis as described above.

Indirect Staining Procedure

1-3. Process samples as above using a working dilution of unlabelled antibody.

4. Resuspend cell pellet first in 50 microliters of HAB for approximately 1 min, then add 50 microliters of a working dilution of the fluorochrome-conjugated second antibody.

5. Vortex briefly and incubate for 20 min at 4°C in the dark.

6. Wash once with 2 ml of PBS-BSA by centrifugation at 250g for 5 min.

7. Resuspend samples in 1 ml of PBS-BSA pre-warmed to 37°C.

8. Proceed with staining for apoptosis as described above.

Note: do not use HAB for staining of immunoglobulin chains. Whenever available, use a monoclonal antibody and/or a reagent directly conjugated to a fluorochrome to minimize unspecific binding. Always use isotypic controls of the same heavy chain class as your relevant antibody for determination of background staining. For single-color staining with FITC-labeled mAbs PI can be used in combination with HO342. For dual-color staining with FITC and PE labeled mAbs 7-AAD has to be used because of the spectral emission overlap of PI and PE.

INFORMATION AND REFERENCES ON DETECTION OF APOPTOTIC CELLS BY FLOW CYTOMETRY

Compared to the classic methods of DNA ladder formation by gel electrophoresis and of morphologic examination by electron microscopy for determination of apoptosis, flow cytometry permits rapid and quantitative measurements on apoptotic cells. Many different flow cytometric methods for the assessment of apoptosis in cells have been described (for a review see 1); most of these methods measure apoptotic changes in cells by staining with various DNA dyes, however, techniques using the terminal deoxynucleotidyl transferase or nick translation assays have also been developed (3). Some of these staining methods utilize unfixed cells (2,4,5,7,8,9,10,11). Due to the fragility of cells undergoing programmed cell death, rapid methods that maintain cells as close as possible to their natural state might be expected to provide the most reliable results. The current rapid 7-AAD staining method uses unfixed cells and thus permits the detection of changes in light scatter parameters and their correlation with other indicators of programmed cell death. However, one drawback of using any live staining method for measuring apoptosis is the variability of dye uptake in different cells and its possible change through certain treatment conditions. Furthermore, reagents which affect membrane permeability (e.g. calcium ionophores) cannot be used with this technique.

Many staining methods for flow cytometry use either fixed cells or treat cells with a hypotonic solution to permit DNA staining by non-vital dyes. The apoptotic cells with degraded DNA appear as cells with hypodiploid DNA content and are represented in so-called "sub-G1" peaks on DNA histograms (6,12). Telford et. al. (12) showed on murine thymocytes that many different DNA dyes produce similar distributions of "sub-G1" peaks irrespective of their binding mode when the same cell fixation method was used. However, when we tried to apply the hypotonic PI method by Nicoletti et al. (6) to human thymocytes our results indicated that use of dissimilar sample preparation methods (e.g. live staining with HO342 vs. hypotonic PI) can lead to dramatic differences in the ability to detect apoptotic cells. Under certain conditions, the treatment of human thymocytes with hypotonic citrate solution containing detergent may not permit leakage of the low molecular weight DNA out of apoptotic cells; this leakage has been proposed as a pre-requisite for the formation of "sub-G1" peaks (1). In murine thymocytes, however, the hypotonic PI staining method produces a clear distinction between live and apoptotic cells (6).

Recently, it has been postulated that the stability and the kinetics of intermediate cell death stages may determine the resistance of a particular cell type to DNA degradation. Consequently, the visibility of "sub-G1" peaks by flow cytometry and also the formation of characteristic DNA ladders on agarose gels might be a function of individual cellular homeostasis. The absence of a "sub-G1" peak on a DNA histogram should not constitute proof of no apoptosis (1). Conversely, it has also been stated that the mere appearance of a hypodiploid DNA peak should not be taken as definite evidence for the presence of an apoptotic cell population without other supporting information (7). Flow cytometric findings in a particular cell type exposed to a certain stimulus must therefore be always verified with other non-flow cytometric methods. Recently, rapid flow cytometric staining methods that use Annexin V for detection of phosphatidylserine exposure on the cell surface as a marker of apoptosis have become commercially available. For this staining method it is essential to add a dead cell discrimination dye like propidium iodide or 7-amino-actinomycin D to the stained cells, because late apoptotic or necrotic cells also express phosphatidylserine and have to be distinguished from the early apoptotic cells by fluorescence (13). The newest flow cytometric assays measure Caspase-3 activity, an early marker of cells undergoing apoptosis and kits for performing this assays are commercially available (14).

Please keep in mind that not all methods for detection of apoptosis in cells may be equally sensitive and techniques must be assessed critically with respect to their applicability to a particular cell type or system.

The following reference list is by no means complete, but should provide a starting point for background information related to the measurement of apoptosis by flow cytometry.

1. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F: Features of apoptotic cells measured by flow cytometry. Cytometry 13:795-808, 1992.

2. Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH: Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Bioch Biophysica Acta 1133:275-285, 1992.

3. Gorczyca W, Gong J, Darzynkiewicz Z: Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951, 1993.

4. Gougeon ML, Garcia S, Guetard D, Olivier R, Dauguet C, Montagnier L: Apoptosis as a mechanism of cell death in peripheral blood lymphocytes in HIV-1 infected individuals. In: Immunodeficiency in HIV infection and AIDS, Janossy G, Autran B, Miedema F (eds.), Karger, Basel, 1992, pp 115-126.

5. Lyons AB, Samuel K, Sanderson A, Maddy AH: Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry. Cytometry 13:809-821, 1992.

6. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth 139:271-279, 1991.

7. Ormerod MG, Collins MKL, Rodriguez-Tarduchy G, Robertson D: Apoptosis in Interleukin-3 dependent haemopoietic cells: quantification by two flow cytometric methods. J Immunol Meth 153:57-65, 1992.

8. Schmid I, Uittenbogaart CU, Giorgi JV: A sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15:12-20, 1994.

9. Schmid I, Uittenbogaart CH, Keld B, Giorgi JV: A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J Immunol Meth 170:145-157,1994.

10. Swat W, Ignatowicz L, Boehmer B, Kisielow P: Clonal deletion of immature CD4+CD8+ thymocytes in suspension culture by extrathymic antigen-presenting cells. Nature 351:150-153, 1991.

11. Swat W, Ignatowicz L, Kisielow P: Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry. J Immunol Meth 137:79-87, 1991.

12. Telford WG, King LE, Fraker PJ: Comparative evaluation of several DNA binding dyes in the detection of apoptosis associated chromatin degradation by flow cytometry. Cytometry 13:137-143, 1992.

13. Vermes I, Haanen C, Steffens-Nakken H, Reutlingsperger C: A novel assay for apoptosis, Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immun. Meth. 184: 39-51, 1995.

14. Nicholson DW, Ali A, Thornberry NA: Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43, 1995.

PROCEDURAL SUGGESTIONS FOR SURFACE ANTIGEN STAINING OF CELLS FOR FLUORESCENCE ACTIVATED CELL SORTING

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I. SAMPLE (one or more of the following preparations)
A. Suspensions of single cells from tissues
B. Tissue culture cells
C. Ficoll-hypaque separated mononuclear cells

II. MATERIALS

A. Antibodies

1. Primary antibodies: directly labeled with a fluorochrome.

2. Secondary antibodies: fluorochrome-labeled Streptavidin or antisera.

B. 1 X PBS + 2% newborn calf serum + 0.1% sodium azide (buffer)

C. Refrigerated centrifuge.

OPTIONAL:
A. Ammonium chloride lysing solution (see attached preparation method and procedure)

B. RPMI 1640 media containing 10% or 20% serum

Staining procedure

1. Place up to 30 X 106 Ficoll-hypaque separated cells in a 12 X 75 mm polypropylene sample tube and wash them once with buffer.

2. Add 0.5 ml of buffer to the pellet; then add the appropriate amounts of the monoclonal antibodies, e.g., FITC, PE, biotinylated or unconjugated antibodies. Usually 1/5 to 1/4 of the amount recommended by the manufacturer per cell number is sufficient for staining.

3. Vortex briefly and incubate for 30 min at 4°C or for 15 min at room temperature in the dark.

4. Wash once with 3.5 ml of buffer by centrifugation at 250g for 10 min. Aspirate supernatant. Resuspend the pellet in 3 ml of buffer.

5. If you need to add a second step reagent, add 150 microliters of buffer to the pellet, then add the appropriate amount of labeled reagent (for avidin, usually 1/4 of the recommended amount is sufficient). Vortex briefly and incubate for 20 to 30 min at 4°C.

6. Wash once with 3.5 ml of buffer by centrifugation at 250g for 10 min, then aspirate supernatant and resuspend in 3 ml of buffer.

Note: if you have more than 30 X 106 cells to stain, use separate tubes for staining of 30 X 106 each. Also, do not start to stain all cells at the same time, wait with the second tube until the first one is done. Thus, the intensity of staining will not fade until all your cells are sorted. For staining of less cells, scale down the reagent and the buffer, e.g., to stain 10 X 106 cells use ~170 microliters of buffer and the appropriately scaled-down amount of antibodies. Resuspend your cells routinely at an approximate concentration of 10 X 106 for the actual sort. For cell preparations that tend to clump excessively, a cell concentration of approximately 5 X 106 can reduce clumping.

It is possible to use RPMI 1640 that contains serum as staining buffer. Sodium azide is not needed as long as the incubation and the washes are done at 4°C. However, we have found that RPMI can increase autofluorescence of cells and is not necessary for good viability (at least for human lymphocytes). Do not use RPMI for staining with avidin, because the biotin it contains will bind all the avidin.

If your cell preparation visually contains residual red blood cells you have to lyse these red cells with ammonium chloride according to the attached protocol. Excessive red cells can interfere with the purity of your sort and can appear in the sorted population.

For a sterile sort it is necessary that staining is done under sterile conditions. If you can use unopened bottles of antibodies, they do not need to be filter-sterilized. If you have to filter your reagents, use 0.22 micron Millipore disk filters that fit on small syringes and minimize your reagent loss. However, you will still loose some of your reagent.

AMMONIUM CHLORIDE LYSING SOLUTION – 10X
Reagents Amount
Ammonium Chloride, ACS 82.9 g
Potassium Bicarbonate, USP 10.0 g
Ethylenediamine tetraacetic acid (EDTA) disodium salt 0.37 g
Water, glass distilled qsad 1.0 liter

Adjust pH to 7.2 and keep in tightly closed container at 4°C. To prepare a 1X working solution (to be used at room temperature), dilute 10X 1:10 with glass distilled water. Keep tightly closed and discard at the end of the day.

LYSING PROCEDURE FOR LYSIS OF RESIDUAL RED BLOOD CELLS

IN FICOLL-HYPAQUE SEPARATED MONONUCLEAR CELLS

1. After the staining procedure, take off as much of the washing buffer as possible without disturbing the pellet.
2.
3. Vortex briefly and add 1 ml of room temperature 1X NH4Cl lysing solution to your cells, vortex again and expose your cells to it for 2-3 minutes at room temperature. Note: do not exceed this time. Centrifuge for 5 minutes at 200g. Remove the lysing buffer completely and resuspend in BUFFER or 1% formaldehyde solution for analysis.

QUESTIONS AND ANSWERS ABOUT CELL SORTING

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Q 1: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations?

A: a) When very high purity (95%-100%) of the target population is required.

b) For separation of populations that have a low density of receptors on their surface.

c) For enrichment of populations on the basis of surface receptor density.

d) For separations on the basis of multicolor staining.

e) For separations on the basis of internal staining e.g of DNA or of internal antigens.

Q 2: Will my cells be harmed by the sorting process?

A: Generally, the cells will be not harmed through the process itself as long as they are maintained continually at a temperature, pH, and in media that is most suited to them.

Q 3: How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells?

A: 1 X 106 = 10 % target population x 50% recovery x 20 X 106 starting cell number. 50% recovery is a reasonable number, but the actual percentage of cells that are recovered with a particular sort depend on a multitude of factors:

a) Cell death that occurs pre- and post sort and loss through adherence of cells to tube walls.

b) Sort rate; the higher the sort rate the lower the recovery.

c) Precision of sort set up of the flow cytometer.

d) If it is an enrichment sort or a purity sort. Enrichment sorts have higher recovery than purity sorts.

Q 4: Are there ways to improve sort recovery?

A: Yes.

a) Use polypropylene tubes for cell processing and sorting.

b) Count cells immediately prior to sorting (after all washes).

c) Use blank Hank’s instead of PBS as sheath fluid.

d) Use polypropylene collection tubes and fill them > 1/3 with media that contains 20% serum.

e) Find optimal temperature for sort (4°C, 15°C, RT).

f) Invert tubes every hour during the sort.

g) Process collection tubes immediately after they are filled up or the sort is finished.

h) Spin collection tubes ~ 10 min.

Q 5: Do I have to use the monoclonal antibodies for staining of the surface antigens at the same cell to reagent ratio as for small cell numbers?

A: Our experience suggests that incubating 20 to 30 million cells in a volume of 0.5 ml of buffer with a quarter to a fifth the amount of antibody that one would calculate as the correct amount through scaling up is sufficient for adequate staining.

Q 6: How long does an average sort take?

A: The average sort takes about 1 hour of set up time for the instrument, up to 15 minutes for setting sort regions for the cells, and 30 minutes of post-sort analysis. Sorting of 20 X 106 cells takes about 2 to 4 hours. The actual time however, is highly dependent on the percentage of the desired cells in the sample as well as the purity and the number of cells that the investigator requested for the post sort assay. The set up time of the flow cytometer for a sterile sort is approximately 90 minutes.