Extrusion of GSH has been reported to be a key event in the apoptotic process (14–17). The cellular thiol-disulfide status is mainly determined by the level and oxidation status of GSH. Thus, the thiol-reacting probe VitaBright-48 may be used for assaying apoptosis. In order to explore the possibility for using VitaBright-48 for assaying apoptosis, we have investigated the correlation of the fluorescence intensity of VitaBright-48 staining with well-known apoptotic markers such as phosphatidylserine externalization and caspase 3/7 activity. During apoptosis, phosphatidylserine is translocated to the outer layer of the membrane, where it may be recognized and bound by the phosphatidylserine selective protein annexin V. Annexin V will also bind to phosphatidylserine on late apoptotic and necrotic cells; however, necrotic cells lack membrane integrity, and thus, necrotic cells can be distinguished from early apoptotic cells by the use of an impermeant dye. Here, we have used staining with CF-647 conjugated annexin V together with the impermeant dye SYTOX green and VitaBright-48. In Figure 2, Jurkat and WEHI-S cells triple stained with VitaBright-48, CF-647-conjugated annexin V, and SYTOX green are shown. Cells that are apoptotic (stained with CF-647 conjugated annexin V) or nonviable (stained with SYTOX green) or late apoptotic (stained with both CF-647 conjugated annexin V and SYTOX green) exhibit a much lower VitaBright-48 fluorescence intensity, indicating a lower level of reduced thiols (Fig. 2 expanded to show all channels as single channels can be found in Supporting Information as Fig. S2). To investigate the timing of the decrease in reduced thiols versus apoptosis induction, cells were induced to undergo apoptosis and were analyzed 5, 10, 15, and 20 h postinduction using image cytometry. The scatter plots in Figure 3 show that control cells exhibit a much stronger VitaBright-48 fluorescence intensity than cells induced to undergo apoptosis. SYTOX green positive cells, that is, nonviable cells, have been excluded from the scatter plot; however, these cells exhibit very low or none VitaBright-48 fluorescence intensity. Already at the first time point (5 h), there is minor decrease in the VitaBright-48 fluorescence intensity and an increase in annexin V-positive cells of CPT-treated Jurkat cells and TNF-α-treated WEHI-S cells compared with the control. Only very few cells exhibit low VitaBright-48 and low annexin V staining, suggesting that the decrease in reduced thiols either happens simultaneously with phosphatidyl serine externalization or after. After 10 h, the percentage of annexin V-positive cells has increased and so has the subpopulation of cells with low VitaBright-48 staining and high annexin V conjugate staining 15–20 h after apoptosis induction using either TNF-α (WEHI-S cells) or CPT (Jurkat cells) the cells are divided into two main subpopulations; one which has a high VitaBright-48 fluorescence intensity and exhibit low staining with the CF-647 annexin V conjugate and one which shows low VitaBright-48 fluorescence intensity but are intensely stained with the CF-647 annexin V conjugate (Fig. 3). There is also an intermediate subpopulation, which shows both high VitaBright-48 fluorescence intensity and high staining with the CF-647 annexin V conjugate. Early after induction of apoptosis (5–10 h), this population is predominant over the low VitaBright-48/high annexin V population; however, later (15–20 h) the population moves to the left; showing a weaker staining with VitaBright-48. This suggests that the apoptosis-mediated decrease in cellular reduced thiols occurs after externalization of phosphatidylserine.
The correlation between VitaBright-48 and caspase 3/7 activity was also investigated. To measure the activity of caspases, the fluorochrome-labeled inhibitors of caspases (FLICA) assay was used (18). Only cells with active caspases will be stained, and thus, it is not necessary to include a nonviable stain; cells lysed by nonapoptotic means will not be stained with the FLICA probe. In Figure 2, Jurkat and WEHI-S cells stained with VitaBright-48 and the red-fluorescent sulphorhodamin-labeled caspase probe; SR-DEVD-FMK are shown. Cells, which are apoptotic (stained with the SR-FLICA probe), exhibit a much lower VitaBright-48 fluorescence intensity than the nonapoptotic cells, indicating that these cells have a lower level of reduced thiols. Figure 4 shows scatter plots of Jurkat and WEHI-S cells stained with VitaBright-48 and SR-FLICA probe. Cells that stain with the SR-FLICA probe and hence are apoptotic stain weakly with VitaBright-48 indicating that the apoptotic cells have a lower level of reduced thiols. As seen from Figure 4, the timing pattern is analogous to what is seen with annexin V staining; first (5–10 h postapoptosis induction), a subpopulation is stained with the SR-FLICA probe whereof some, but not all, also have a low VitaBrigh-48 fluorescence intensity and then, 15–20 h after induction the SR-FLICA-stained subpopulation exhibit a more pronounced decrease in VitaBright-48 staining. This suggests that the most dramatic decrease in thelevel of reduced thiols happens after activation of caspases 3 and 7.