For PP2 assay, day 6–10 blasting T cells were pre-incubated with PP2 (10 μM; 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Sigma-Aldrich) for 1 h at 37 °C and 5% CO2. Pre-treated cells were imaged at 37 °C and 5% CO2 in the presence of 10 μM PP2.
For CD4 and CD28 blockade, an anti-CD4 (clone Gk1.5, BioLegend) or anti-CD28 (clone 37.51, BioLegend) Fab was prepared and purified with Micro Fab Preparation Kit (Thermo Fisher Scientific). Day 6–10 T cells were pre-incubated for 1 h at 37 °C and 5% CO2 with anti-CD4 Fab (2 μg/mL) or anti-CD28 Fab (4 μg/mL) then imaged in the presence of 2 μg/mL Fab.
For CD4 and CD28 blockade, an anti-CD4 (clone Gk1.5, BioLegend) or anti-CD28 (clone 37.51, BioLegend) Fab was prepared and purified with Micro Fab Preparation Kit (Thermo Fisher Scientific). Day 6–10 T cells were pre-incubated for 1 h at 37 °C and 5% CO2 with anti-CD4 Fab (2 μg/mL) or anti-CD28 Fab (4 μg/mL) then imaged in the presence of 2 μg/mL Fab.