Early Apoptotic Cells Detected by 7-Aminoactinomycin and Annexin V Are Positive for Fluorescence Tagged Activated Caspase-9 Inhibitor (Red-LEHD-FMK)

7-Aminoactinomycin (7AAD) and annexin V (AnnV) are commonly used in flow cytometry for the detection of apoptosis. Use of 7AAD/AnnV allows cells to be distinguished between viable, early and late/dead apoptotic cells through different staining populations. Cells stained negative for 7AAD were shown to contain a population of AnnV⁺ cells, implicating cells in early apoptosis (Hasper, H. J., Weghorst, R. M., Richel, D. J., Meerwaldt, J. H., Olthuis, F. M., & Schenkeveld, C. E. (2000). Cytometry 40, 167– 171). We show here that the 7AAD⁻/AnnV⁺ population displays caspase-9 (C9) activation. Jurkat E6.1 human leukaemic T cell lymphoblast were induced into apoptosis by treating with 0.1 μg/ml CD95 ligand antibody (CD95ab), 5 μg/ml cycloheximide (CLX) or 2 μg/ml camptothecin (CPT) for 16 hours. C9 activation was detected using a phycoerythrin (PE/RED) tagged specific inhibitor of active C9, LEHD-FMK (Red-LEHD-FMK). Treated and control untreated cells were incubated with Red-LEHD-FMK, washed and followed by 7AAD and AnnV. Activated caspase-3 (C3) staining was conducted with anti-active C3 antibody following AnnV. Cells were analysed by flow cytometry. Cells, induced into apoptosis by each of the 3 different treatments, showed 4 different population groupings –

1. 7AAD⁻/AnnV⁻/C9⁻,

2. 7AAD⁻/AnnV⁺/C9⁺,

3. 7AAD^+low/AnnV⁺/C9⁺ and

4. 7AAD^+high/AnnV⁺/C9⁺, possibly implicating

1. live,

2. early,

3. mid and

4. late/dead apoptosis respectively.

In addition, in the assay of C3 activation (active caspase-3 antibody) and AnnV staining, the majority of AnnV⁺ cells showed active C3. A time course (0, 2, 4, 6, 8, 10, 12, 14 and 16 hours) of apoptosis induction, followed by 7AAD/AnnV/C9 staining, showed that viable cells (7AAD⁻/AnnV⁻/C9⁻) decreased from 85% at 0 hours to 3%, 12%, 22% (CD95ab, CLX and CPT treatment, respectively) at 16 hours. The early apoptotic (7AAD⁻/AnnV⁺/C9⁺) population decreased after initial treatment from 61% at 2 hours to 36% at 16 hours in CD95ab treated Jurkat cells, whereas in CLX and CPT treated Jurkat cells, this population decreased from ~44% at 2 hours to ~38% at 16 hours. The mid apoptotic (7AAD^+low/AnnV⁺/C9⁺) population remained fairly constant from 8 hours of induction (between 10–22%) after all treatments. The late/dead apoptotic (7AAD^+high/AnnV⁺/C9⁺) population increased after CD95ab treatment from 3% at 2 hours to 39% at 16 hours, and after CLX and CPT treatment, from ~4% at 2 hours to ~28% at 16 hours. These results show that the early apoptotic population detected by 7AAD and AnnV (7AAD⁻/AnnV⁺) also exhibit activated C9, and possibly activated C3. This early apoptotic (7AAD⁻/AnnV⁺/C9⁺) population is high early after treatment at 2 hours, but with time, decreases and becomes either mid apoptotic (7AAD^+low/AnnV⁺/C9⁺) or late/dead apoptotic (7AAD^+high/AnnV⁺/C9⁺). In summary, the 7AAD negative population does not represent viable cells only, but also contains cells displaying AnnV⁺, and C9 and C3 activation. Although each of the 3 inducers of apoptosis eventually results in cell death; the rate of apoptosis, as detected by 7AAD/AnnV/C9 staining, varies, possibly due to different signalling pathways selective for each inducer. The sequence of events in the apoptosis pathway needs to be further delineated using the described and other markers of apoptosis/cell death.