Apc labeled mouse igg1 isotype control mab clone mopc 21

Human CAR-T cells were incubated with Human TruStain FcX (Biolegend, San Diego, CA, USA) and then stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) and with PE-Cy7-labeled anti-CD8α mAb (clone HIT8α, Biolegend, San Diego, CA, USA). For CAR expression analysis based on binding to VEGFR2 protein, cells were further stained with recombinant human VEGFR2/KDR Fc chimera protein (VEGFR2-Fc, R&D Systems, Minneapolis, MN, USA) and APC-labeled anti-human IgG Fc mAb (clone HP6017, Biolegend, San Diego, CA, USA). For CAR expression analysis using HA-tag detection, cells were further stained with APC-labeled anti-HA-tag mAb (clone 16B12, Biolegend) or APC-labeled mouse IgG1 isotype control mAb (clone MOPC-21, Biolegend, San Diego, CA, USA). Cells were resolved using BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) or Gallios (Beckman Coulter, Brea, CA, USA) flow cytometers, and data were analyzed using FlowJo software v10 (FlowJo LLC, Ashland, OR, USA). Data were represented as GMFI ratios, calculated according to the following formula: GMFI ratio = (GMFI of CAR-T cells stained with anti-HA mAb or anti-human IgG Fc mAb with VEGFR2-Fc)/(GMFI of CAR-T cells stained with mouse IgG1 isotype control mAb or anti-human IgG Fc mAb without VEGFR2-Fc).

Murine CAR-T cells were incubated with an anti-mouse CD16/CD32 mAb (clone 93, Biolegend, San Diego, CA, USA), and then stained with the Zombie Aqua Fixable Viability Kit (Biolegend) and PE-Cy7-labeled anti-CD8α mAb (clone 53-6.7, Biolegend). CAR expression was evaluated using APC-labeled anti-HA.11 Epitope Tag mAb (clone 16B12, Biolegend) or APC-labeled mouse IgG1 isotype control mAb (clone MOPC-21, Biolegend). Immunofluorescence was measured using a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Data were represented as GMFI ratios, calculated according to the following formula: GMFI ratio = (GMFI of CAR-T cells stained with anti-HA mAb)/ (GMFI of CAR-T cells stained with mouse IgG1 isotype control mAb).