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Use the Total Cytotoxicity Test (TCT) for an accurate, quantitative assessment of cytolytic activity due to apoptosis and necrosis. The TCT assay does not use any radioisotopes, making it much safer and more cost effective to run than 51Cr assays. It provides a direct measurement of cytotoxicity rather than using an indirect indicator, such as the release of LDH or ATP enzymes, and there is no need to lyse the cells.

The Total Cytotoxicity Test Kit (TCT) contains three fluorescent reagents for the analysis of cell-mediated cytotoxicity and apoptosis. The first, carboxyfluorosuccinimide ester (CFSE), is a green fluorescing cellular protein stain used to identify the target cells in the cytotoxicity assay. The second reagent is 7-aminoactinomycin D (7-AAD), a red live/dead viability stain, used to identify late apoptotic and necrotic target cells killed in the cytotoxicity assay. The third reagent is ICT''s orange-red FLICATM reagent, SR-VAD-FMK. This fluorescent probe measures caspase activity in the target cell population, allowing the user to identify caspase-positive cells in the early stages of apoptosis that would otherwise be missed from an analysis that identifies only necrosis.

Using the TCT assay, all of the target cells are initially labeled with CFSE. The unstained immune effector cells, cytotoxic drug compounds, or antibodies + complement can then be added and incubated for the desired length of time. Following the induction period, the SR-FLICA reagent is incubated with the cells to identify those containing active caspases. Finally, 7-AAD is added to stain all of the dead cells red, following which, the cells are ready for flow cytometric analysis. By initially gating on the green (CFSE+) target cells only, a two parameter dot plot will reveal target cells that are completely healthy (green only) from those that are in early apoptosis (green and orange-red), late apoptosis (green, orange-red, and red) and necrotic (green and red).