Fitc cd3 antibodies

Surface protein expression was determined by flow cytometry. After electroporation for 5 days, 1×10⁶ cells were incubated with APC-CD4, PE/Cy7-TCR (or PE-TCR) and FITC-CD3 antibodies (Biolegend) for 30 mins. For the CD22BBz CAR transduced T cells were incubated with 0.2 μg CD22-Fc (R&D system) in 100 μL PBS for 30 mins, and then stained with PE-IgG-Fc (Biolegend). For the CD19BBz CAR detection, the transduced T cells were stained with APC-anti-DYKDDDDK Tag (Biolegend). Stained cells were measured and sorted on BD FACSAria II. For the T cell exhaustion assay, T cells from various groups were co-cultured with NALM6 cells at 0.5:1 E:T ratio for 24 hours. 1×10⁶ cells were incubated with 0.2 μg CD22-Fc (R&D Systems) in 100 μL PBS for 30 mins and then stained with PE-IgG-Fc, PD-1-FITC, TIGIT-APC and LAG3-Percp/cy5.5 (Biolegend) for 30 mins. After washing twice, the stained cells were measured and sorted on BD FACSAria II, and analyzed using FlowJo software version 9.9.4 or 10.3 (Treestar, Ashland, OR).