Sytox aadvanced dead cell stain solution

A Cell Trace CFSE (Carboxyfluorescein succinimidyl ester) Cell Proliferation Kit (Invitrogen, Carlsbad, CA, USA) was used to label the resting T cells. Cells were washed with PBS, resuspended in 5 µM CFSE solution in 1 ml phosphate buffered saline (PBS) containing 5% fetal calf serum (FCS) and incubated for 5 min at room temperature in the dark. Then cells were washed twice in 10 ml PBS containing 5% FCS to remove excess CFSE. Stained T cells were resuspended in AIM-V culture medium at a concentration of 1 × 10⁶ cells/ml and seeded in anti-CD3/CD28 antibody coated 24-well plates (Corning Inc., Amsterdam, The Netherlands). To investigate the effect on freshly stimulated T cells, increasing concentrations of ADV, Cladribine or DMSO as vehicle control were added in parallel at the beginning of the experiment. In addition, to study the effect on pre-activated T cells, compounds were added 48 h after stimulation. After an incubation time of 96 h, cells were transferred to polystyrene tubes, washed with FACS staining buffer and incubated with 1 µl of SYTOX AADvanced dead cell stain solution (Thermo Fisher Scientific, Waltham, MA) for 5 min to exclude dead cells. Cells were analyzed by flow cytometry (LSRFortessa, BD Biosciences, Franklin Lakes, NJ, USA).

Target cells were pre-stained with CellTrace™ Violet (Thermo-Fisher Scientific, C34557) at a final concentration of 5 μM in PBS for 15 min at 37°C. After staining, the cells were washed in complete culture medium prior to being mixed with NK cell cultures at the indicated effector to target (E:T) ratios. After a brief centrifugation, co-cultures were incubated at 37°C for 3.5 h. Afterwards, CellEventR Caspase-3/7 Green Detection Reagent (Thermal Fisher Scientific, C10423) was added for an additional 30 min of culture for a total incubation time of 4 h. During the final 5 min of staining, SYTOX™ AADvanced™ dead cell stain solution (Thermal Fisher Scientific, S10349) was added and mixed gently. Cells were then analyzed by flow cytometry.