Anti cd3 cd28 antibody coated 24 well plates

For measurement of cytokine secretion, human T cells (10⁶ cells/ml) were seeded in anti CD3/CD28 antibody coated 24-well plates (Corning Inc.) and treated with increasing concentrations of ADV or vehicle control. Supernatants were harvested after 72 h and cytokine concentrations (IFN-γ, IL-5, IL-10, and IL-17) were determined with specific ELISA (Bio-techne Ltd., Minneapolis, MN, USA) according to manufacturer’s instructions. The optical density of each well was determined at 450 and 570 nm (Tecan Safire Microplate Reader, Tecan Group, Männedorf, Switzerland). The concentration of each cytokine was calculated using a standard curve and the measured absorbance.

For measurement of cytokine secretion, human T cells (10⁶ cells/mL) were seeded in anti-CD3/CD28 antibody-coated 24-well plates (Corning Inc., Amsterdam, The Netherlands) and treated with increasing concentrations of Pitavastatin or vehicle control. Supernatants were harvested after 72 h, and cytokine concentrations (IFN-γ, IL-5, IL-10 and IL-17) were determined with specific ELISA (bio-techne Ltd., Minneapolis, MN, USA), according to manufacturer’s instructions. The optical density of each well was determined at 450 and 570 nm (Tecan Safire Microplate Reader, Tecan Group, Männedorf, Switzerland). The concentration of each cytokine was calculated by using a standard curve and the measured absorbance (n = 4 independent experiments).

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).