Commonly used Cytometry Apoptosis Detection Techniques

Hoechst and 7-AAD

H342 is DNA stain that binds preferentially to A-T base-pairs without the necessary of permeabilization step. However, it is necessary to set up incubation conditions for efficient internalization of the dye. Hoechst 33342 (Bisbenzimidazole, Sigma B-2262) and 33258 can be used for FACS analysis of apoptosis. 7-AAD (7-Amino-actinomycin, Sigma A-9400) is used for identification of necrotic and late apoptotic cells.



Controls:

No treatment no stains cells control

Treated cells with Hoechst only

Treated cells with 7-AAD only

Untreated cells with both stains



Procedure:

- Resuspend at least 0.5 x 10^6 cells in 12x75mm tubes at a concentration of 10^6 cells/ml.

- 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.



Reference: http://biochem.dental.upenn.edu/~sdmfacs/apopproc.htm



TUNEL

TUNEL (TdT-mediated dUTP Nick End Labelling) assay is commonly used to detect DNA fragmentation by labeling 3’OH termini of DNA fragments with biotin-conjugated nucleotides. TUNEL uses the enzyme terminal deoxynucleotidyl transferase (TdT) to label cells that have oligonucleosomal nicks/strand breaks and 3’OH termini exposed in their DNA.



Annexin

It has been shown that phosphatidylserine (PS) is exposed on membrane surface in apoptotic cells. The apoptotic cells can further expose the internal PS in vitro during membrane bound apoptotic bodies formation. Annexin-V (AV) / FITC conjugate can deted PS externalization during apoptosis. Staining is normally done in combination with PI or 7-AAD to detect the late apoptotic and necrotic cells. Annexin V-7-AAD staining can detect live viable cells (AV - / 7-AAD -), early membrane intact (AV + / 7-AAD -) and late apoptotic or necrotic cells (AV and 7-AAD +).

Archived page

Last update 19-Mar-2008, Rating n/a of 0 votes.