Comparison of three thiol probes for determination of apoptosis-related changes in cellular redox status

An early step in apoptosis is extrusion of reduced glutathione (GSH). Current assays for measuring apoptosis involve a number of incubation and washing steps, making them time consuming and laborious. Using two novel thiol reactive agents (VitaBright-43 and VitaBright-48) and a GSH specific probe; monochlorobimane, we investigated whether changes in the level of free thiols can be used as an apoptotic marker. Upon addition to cells the probes permeate the cell membrane and react with intracellular thiols, causing cellular fluorescence. Cytometric quantification of the cell fluorescence (without washing) can then be used to determine the population's cellular thiol level at the single cell level. Apoptotic traits such as phosphatidylserine externalisation, caspase activity and mitochondrial potential were investigated at different time points after induction of apoptosis and correlated to changes detected using the thiol probes. We found that though all three thiol probes could be used to detect changes in the level of free thiols correlating well with apoptotic markers, other properties such as detection of early versus late apoptosis and staining kinetics differed among the three probes. However, we suggest adding evaluation of the level of free thiols to the list of phenotypes which may be measured in order to detect apoptosis, as this provides a reliable and easy way of assaying apoptosis. © 2013 International Society for Advancement of Cytometry