There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis, e.g. Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9 and Caspase 10. The functions of these enzymes are not yet entirely clear, but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and execution of the apoptotic cascade. In classic ealry and late apoptosis (as opposed to RIP1-dependent apoptosis) when activated caspase-3 is expressed Jurkat cells become RIP3 negative in live or dead cells respectively. Because of the central importance of the caspases in apoptosis, their detection by flow cytometry has become widespread. The activity of enzymes implicated in apoptosis may be detected in three ways:

• By detecting the active form of the enzyme using a specific antibody. Various forms of cell death e.g. Oncosis, Apoptosis & Necroptosis display various levels of Caspase-3, see figure
• ER stress only displays background levels of Caspase-3 activiyt, see figure
• Addition of fixable viability dyes, RIP3 and Caspase-3 allows the detection of early and late apoptosis by flow cyometry, see figure.
• By using a fluorochrome labelled inhibitor peptide that binds to the active site of the caspase or FLICA - Fluorescent Labelled Inhibitor Caspase. Invitrogen kit V35117 includes FAM-VAD-FMK poly caspases, Propidium Iodide and Hoechst 33342 for live and dead cell imaging. The Hoechst supplied in this kit can be used for live cell cell cycle analysis to determine if caspase activation is cell cycle dependent
• By using a non-fluorescent substrate for the enzyme which yields a fluorescent product if the enzyme is active - eg PhiPhiLux^® (http://phiphilux.com/phi1.html [new window])