FACS Procedures for Apoptosis Detection

  1. Hoechst vs. 7-AAD
  2. TUNEL
  3. Annexin V vs. 7-AAD

NOTES:  We use these protocols with PBMC.  It may be necessary to fine-tune them for use with other cell types.  In all cases, samples should be brought in 12x75mm snap cap tubes (Falcon 2054) at a final concentration of 106 cells/ml and a minimum volume of 0.5ml.

Hoechst vs. 7-AAD

NOTES:  It is possible to perform this assay with Hoechst 33342 (Sigma B-2262), but the increase in fluorescence seen in the apoptotic cells may be less dramatic.  We use log. amplification with 33258 and linear with 33342.  7-AAD is included for identification of necrotic/late apoptotic cells.  PI could be substituted.  Hoechst dyes can also be obtained from Molecular Probes.

Materials:

Controls Required: 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.
  3. Incubate the samples in a 370 waterbath for 7 minutes.
  4. Remove samples from the waterbath and immediately place on ice.
  5. Add 7-AAD at a final concentration of 1µg/ml to samples.
  6. Incubate on ice for 10 minutes.
  7. Analyze samples.
FACS setup:

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:

Controls Required: 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:

Controls Required: 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: