Assays for Cell Viability and Death

Jurkat cells in the exponential phase of growth were seeded on the white-walled 96-well microplate wells with transparent bottom (Greiner Bio-One, Frickenhausen, Germany), 2 × 10⁴ cells per well in 100 μl of medium. Appropriate toxin and tetrapeptide were added to the cells at the same time and maintained for indicated time periods at 37°C, 5% CO₂. As a positive control, pan-caspase inhibitor Q-VD-OPh (5 μM) was added to the toxin-treated cells. The positive control treatment for necroptosis assay included 5 ng/ml of anti-Fas antibody, 5 μM of Q-VD-OPh and 20 μM of necrostatin-1 (Nec-1) (Santa Cruz Biotechnology, Santa Cruz, CA, United States) as a specific inhibitor of necroptosis. Nec-1 was preloaded for 1 h before adding the rest of the agents. As the negative controls, toxin alone or toxin plus inactive tetrapeptide (SKGS) were used.

Cell viability was estimated by measuring the levels of intracellular ATP using CellTiter-Glo reagent (Promega, Madison, WI, United States). CellTiter-Glo was added with the ratio 1:1 (100 μl per well) at the end of each treatment assay. The plate was manually shaked for two minutes to facilitate component mixing and cell lysis. After 15 min of incubation at room temperature (RT) in the dark, the luminescence was measured with Tecan GENios Pro microplate reader (Tecan Group, Switzerland).

In some experiments the extent of cell death was measured in addition to the cell viability. To measure the amount of dead cells, 250 nM SYTOX Green nucleic acid stain (Life Technologies, Eugene, OR, United States) that detects the cells with compromised plasma membrane was added to the cells without washing. The cells were then incubated for 15 min at RT in the dark and the fluorescence was measured with Tecan GENios Pro microplate reader using 492 nm excitation and 535 nm emission filters. Cell death assay was performed prior to measurement of ATP levels from the same wells.

To measure the activity of caspase-3/7 the cells were incubated with CH11 antibody and DTT-pretreated peptide (CKGC, dCKGC, or SKGS) or pan-caspase inhibitor Q-VD-OPh for 4 h at 37°C, 5% CO₂. Caspase-Glo 3/7 reagent (Promega, Madison, WI, United States) was added with ratio 1:1 (100 μl per well) and incubated for 30 min at RT in dark before the luminescence was measured with Tecan GENios Pro microplate reader. Caspase-8 activity was measured by similar procedure, except that the cells were incubated with peptides for 3 h and then for 1 h with Caspase-Glo 8 reagent premixed with MG-132 protease inhibitor (Promega).