FACS Procedures for Apoptosis Detection

Materials:

• Hoechst 33258 (Sigma B-2883).

  • stock: 10 mg/ml in dH20 (4⁰)

  • working dilution: 500µg/ml (50µl stock + 950µl PBS).

• 7-Amino-actinomycin (Sigma A-9400)

  • stock: 200µg/ml in dH20 (4⁰)

• 12x75mm snap cap tubes (Falcon 2054).

Controls Required:

• Cells only (no treatment/no stain)

• Treated cells with Hoechst only

• Treated cells with 7-AAD only

• Untreated cells with both stains

Procedure:

1. Resuspend at least 0.5 x 10⁶ cells in 12x75mm tubes at a concentration of 10⁶ cells/ml.

2. Add Ho 33258 at a final concentration of 1µg/ml to samples.

• 2µl/ml of working solution per tube.

Incubate the samples in a 37⁰ waterbath for 7 minutes.

Remove samples from the waterbath and immediately place on ice.

Add 7-AAD at a final concentration of 1µg/ml to samples.

• 5µl/ml of stock solution per tube.

Incubate on ice for 10 minutes.

Analyze samples.

FACS setup:

• Primary laser: 310nm (UV), 50mW.

• Secondary laser: 488nm (visible), 250mW.

• Ho 33258 detection: 424/44 BP filter, Fl-1 detector.

• 7-AAD detection: 660/20 BP filter, Fl-4 detector.

TUNEL

NOTES:  This technique uses the enzyme terminal deoxynucleotidyl transferase (TdT) to label cells that have oligonucleosomal nicks/strand breaks in their DNA.  We use the In Situ Cell Death Detection Kit from Boehringer Mannheim, which uses directly labeled dUTP-FITC.  Other protocols call for BrdU and FITC-anti BrdU, biotinylated dUTP and streptavidin-FITC, etc.  PI or 7-AAD can be included for cell cycle determination, although it is very difficult to obtain tight CVs.  The protocol below is carried out in 96-well V-bottomed micro titer plates (MTP); with some modifications, it could be carried out in 12 x 75 mm plastic tubes.  Phoenix Flow Systems has a protocol for pre-fixing samples with PFA and ethanol, storing them, and labeling them at some later date.

Materials:

• In Situ Cell Death Detection Kit (Boehringer Mannheim 1 684 79550)

  • Set aside 100µl label solution for 2 "no enzyme" controls

  • Add all (50µl) of one bottle of enzyme solution to the remaining label solution (450µl) and mix to make enough reaction mixture for 10 samples

• 96 well V-bottomed microtiter plate (MTP).

• 12x75mm snap cap tubes (Falcon 2054).

• PBS + 1% BSA (Sigma A-7030).

• Fresh 4% paraformaldehyde in PBS pH 7.4.

• Shaker for incubating cells in MTP.

• Permeabilization buffer: 0.1% Triton X-100 in 0.1% sodium citrate.

• Propidium Iodide (PI) buffer (optional)

  • 5µg/ml PI (Sigma P-4170) and 200µg/ml DNase-free RNase A (Sigma R-5503) in PBS.

Controls Required:

• Cells only (no treatment/no stain)

• Cells + label WITHOUT enzyme

• Untreated/non-apoptotic cells with label and enzyme

• If PI has been included: one tube with untreated fixed cells in PI buffer, WITHOUT TUNEL label or enzyme

Procedure:

1. Wash cells twice in PBS/1% BSA at 4⁰.

2. Resuspend to 2x10⁷ cells/ml.

3. Add 100µl cells to one MTP well for each sample and control.

4. Add 100 ul fresh 4% paraformaldehyde in PBS pH 7.4 to each well.

5. Resuspend thoroughly and incubate 30 minutes at room temperature WHILE SHAKING.

6. Centrifuge MTP at 300xG for 10 minutes and remove fixative by flicking or suction.

7. Wash once with 200µl PBS/BSA as above.

8. Resuspend cells in 100µl permeabilization buffer per well for 2 minutes on ice (4⁰.)

9. Wash twice as above.

10. Resuspend each sample in 50µl reaction mixture and the "no enzyme" control in 50µl label solution.

11. Cover MTP and incubate 60 minutes at 37⁰ in humidified air in the dark.

12. Wash twice as above.

13. Resuspend each well in a 12x75mm tube with 500µl PBS/BSA OR with 500µl PI buffer

14. Analyze.

Annexin V + 7-AAD

NOTES:  For identification of necrotic/late apoptotic cells, we substitute 7-AAD for the PI provided with the kit.

Materials:

• Apoptosis Detection Kit (R&D Systems KNX50).

  • contains: FITC-conjugated human annexin V, PI, 10x Binding Buffer.

• 7-Amino-actinomycin (Sigma A-9400)

  • stock 200µg/ml in dH20 (4⁰) .

• 12x75mm snap cap tubes (Falcon 2054).

• PBS.

Controls Required:

• Cells only (no treatment/no stain)

• Treated cells with FITC-Annexin V only

• Treated cells with 7-AAD only

• Untreated cells with both stains

Procedure:

1. Resuspend at least 0.5 x 10⁶ cells in 12x75mm tubes at a concentration of 10⁶ cells/ml.

2. Add 2ml of cold PBS to tubes.

3. Centrifuge for 8 minutes at 1800rpm.

4. Resuspend pellet in 2ml of cold PBS.

5. Centrifuge for 8 minutes at 1800rpm.

6. Resuspend cells in 0.1ml of 1x binding buffer.

7. Add 10µl of FITC-conjugated annexin V and 5µl of 7-AAD to tubes.

8. Gently vortex.

9. Incubate at room temperature for 15 minutes in the dark.

10. Add 0.9ml of 1x binding buffer.

11. Analyze samples within 1 hour of staining.

FACS setup:

• Primary laser: 488nm (visible), 250mW.

• FITC-annexin V detection: 530/20 BP filter, Fl-1 detector.

• 7-AAD detection: 660/20 BP filter, Fl-3 detector.