Several pathways lead to apoptotic cell death through the cleavage of zymogens and the generation of specific proteases called caspases. The caspase family can be subdivided into three classes: initiation, amplification and executioner. These classes are generally defined as follows: i) initiation caspases activated by death receptors (i.e., caspase-8); ii) amplification caspases activated by initiation caspases or mitochondrial molecules (i.e., caspase-9); and iii) executioner caspases activated by upstream caspases (i.e., caspase-3). All caspases are cysteine proteases and cleave their substrates immediately after an aspartate residue.
Activation of executioner caspases serves as an early marker of apoptosis for flow cytometric studies (1)
. However, the fluorogenic substrates needed to analyze these enzymes are not cell permeable. Some antibodies are able to specifically label the cleaved form of caspase-3 and can be used in multivariate analysis to reveal early apoptosis in leukemic cells (2)
. This approach necessitates cell fixation and permeabilization, and it is time-consuming.
The inhibitor peptide, CaspACE™ VAD-FMK In Situ Marker, is bound with varying affinities by most caspases. Thus, a fluorescent, cell-permeable derivative of VAD-FMK is potentially an interesting tool for flow cytometry analysis of the global activation status in the caspase pathway.
CaspACE™ FITC-VAD-FMK In Situ Marker Labeling
CaspACE™ FITC-VAD-FMK In Situ Marker (Cat.# G7461, G7462) was added directly to the cell culture medium (HEPES-buffered RPMI 1640 with 10% fetal calf serum) at a final concentration of 5µM (1µl stock solution in 1ml of medium). After incubation for 20 minutes at 20°C, the cells were centrifuged, washed in 1ml of PBS and fixed in 1ml of 0.5% formaldehyde for 30 minutes at 20°C. The cells were centrifuged again and suspended in 0.5ml of PBS prior to flow cytometry analysis.
Flow Cytometry Analysis
Flow cytometry analysis was performed using an EPICS® XL Cytometer (Beckman-Coulter). Fluorescence was measured at 530nm (excitation of 488nm). Fixation was found to facilitate the discrimination between fluorescent and negative populations. The negative population was defined as the higher population of the untreated sample. However, the experiment described in Figure 1 was performed without fixation, as the intent was to measure the mean fluorescence of the whole population and not to discriminate between positive and negative cells. The cell sorting experiment was performed using an Elite cell sorter (Beckman-Coulter).
Annexin V Labeling
The FITC-annexin V kit (Immunotech, Beckman-Coulter) was used to detect apoptosis as specified by the supplier. Briefly, Jurkat T cells were suspended in Ca2+-rich binding buffer, and 5µl of FITC-annexin V were added. After a 10-minute incubation at 4°C, the cells were analyzed using an EPIC XL cytometer. The fluorescence was measured as described above.
Fluorescence Microscopy Analysis of Apoptotic Cells
The cells were incubated in culture medium with either 1µg/ml acridine orange or 0.5µg/ml Hoechst 33342 (Molecular Probes). Cells with either condensed chromatin or fractionated nuclei were identified as apoptotic by fluorescence microscopy. A minimum of 100 cells were counted for each sample.
To be a good marker of apoptosis, the CaspACE™ FITC-VAD-FMK In Situ Marker must move easily in and out of healthy cells and remain anchored inside apoptotic cells. The experiment described in Figure 1 was performed to verify this property. FITC-VAD-FMK (5µM) was added to Jurkat cells, and the evolution of cell fluorescence was monitored every 4 minutes by flow cytometry. The uptake of the fluorescent marker by the cells was accomplished in less than 4 minutes, after which fluorescence remained stable for 30 minutes (Figure 1). When the cells were washed by centrifugation and suspended in marker-free PBS, a steep decrease in fluorescence was observed, suggesting that greater than 90% of the FITC-VAD-FMK had been washed free from control cells. However, when the cells were pretreated with a Fas-agonist antibody to induce 80% apoptosis, their fluorescence remained three-fold higher than that of the control (Figure 1). These results confirm that the CaspACE™ FITC-VAD-FMK In Situ Marker is cell-permeable and is better retained in apoptotic versus healthy cells.
One of the advantages of the cell-by-cell analysis afforded by flow cytometry is the ability to perform population analysis and thus account for the heterogeneity of samples. Figure 2 shows that the response of Jurkat cells to the Fas-agonist occurred in two-steps. Following FITC-VAD-FMK addition, a small population with low fluorescence intensity was observed. This low fluorescence was also observed in the sample in which apoptosis was not induced (Figure 2, 0 hour). This population increased progressively during 2 hours of treatment with the Fas agonist antibody. After 2 hours of treatment, a population with high fluorescence intensity appeared, and this population increased during the subsequent hour.
Following treatment with the Fas-agonist, the no-, low- and high-fluorescence populations were sorted by flow cytometry, stained with the DNA binding fluorochrome, Hoechst 33342, and observed by fluorescence microscopy (Figure 3). The FITC-VAD-FMK negative cells were clearly identified as intact cells (Panels A–C). The highly fluorescent cells were identified as late apoptotic cells with condensed chromatin and fragmented nuclei (Panels G–I). The low-fluorescence cell population was more heterogeneous and included intact cells (Panel D), early apoptotic cells (Panel E) and late apoptotic cells (Panel F).
The time-course evolution of the percentage of low and high FITC-VAD-FMK labeled cells was monitored by flow cytometry (Figure 4, Panel A). The low-fluorescent cell rate increased first (Figure 4, Panel A) and then remained at a constant level while the high-fluorescence cells began to increase (Figure 4, Panel A). During this time, the total number of apoptotic cells increased continuously (Figure 4, Panel B). The results of these experiments suggest that low-FITC-VAD-FMK-labeled cells are early apoptotic while the highly fluorescent cells are late apoptotic.
The time-course analysis of the whole FITC-VAD-FMK-labeled cell population was also compared with the number of apoptotic cells as measured at the membrane level by flow cytometry or microscopically at the nucleus level (Figure 4, Panel B). A good relation was found between the percentage of positive cells and the rate of apoptosis with both methods, confirming that FITC-VAD-FMK labeled cells were apoptotic. Similar results were obtained during Fas-driven apoptosis in another cell line, U937 (Figure 5). After three hours of Fas agonist treatment, an irreversible caspase inhibitor was added in excess and incubated for 15 minutes before labeling the cells with either FITC-VAD-FMK or annexin V. The addition of the caspase inhibitor decreased the percentage of FITC-VAD-FMK-labeled cells (Figure 5, blue bar) without affecting the percentage of annexin V-labeled cells (green bar). This suggests that active caspases are necessary for apoptotic cells to be labeled with FITC-VAD-FMK.
Finally, the labeling of U937 cells by FITC-VAD-FMK was compared to the rate of annexin V labeling after treatment with different apoptosis inducers (Figure 6). All spontaneous- (C), Fas- (anti-Fas), C2-ceramide-(C2-Cer) and daunorubicine-induced (DNR) apoptosis showed similar levels of fluorescent cells with both labels after 6 hours. This confirms that FITC-VAD-FMK can be used as a marker of apoptosis with flow cytometry, whatever the apoptosis induction method.
As shown here, FITC-VAD-FMK can be used to label apoptotic leukemic cells for flow cytometry analysis. The percentage of apoptotic cells detected using this marker is in agreement with other methods. Interestingly, FITC-VAD-FMK labeling was able to discriminate between early and late apoptosis through fluorescence intensity (Figures 2–4). These intensity changes may be related to the activation of initiation (early) or executioner caspases (late). The FITC-VAD-FMK marker was able to detect apoptosis induced by several molecules, including ceramide, while no caspase-3 activation was detected in this pathway (3)
FITC-VAD-FMK should be a useful tool to reveal caspase-dependent apoptosis by multivariate flow cytometry analyses. A reliable negative control, however, will be necessary for analyzing samples of heterogeneous cell populations such as blood or bone marrow. Currently, nonapoptotic controls are not easily available for these samples types.
Finally, although FITC-VAD-FMK binding can be considered as a good marker of apoptosis, it is true that general caspase activation is not exclusive for apoptosis and can also occur in some inflammatory and differentiation processes (for review, see reference (4)