Cell surface proteins on CAR-T cells were stained with the following antibodies: CD4-APC/H7, CD8a-PE/Cy7, CCR7-PerCP/Cy5.5, CXCR3-AF488, CD69-FITC, CD3-APC (all from BD Biosciences, San Jose, CA, USA); CD8a-FITC, CD28-FITC, CD27-PE, LAG-3-PE, TIM-3-FITC, TIGIT-PerCP/Cy5.5, CD45RA-APC (all from Biolegend, San Diego, CA, USA); PD-1-APC (eBioscience, Thermo Fisher Scientific, San Diego, CA, USA). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY, USA) followed by a BV421-conjugated anti-Fc antibody (Biolegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in 1% (v/v) FCS in PBS or 1% (v/v) paraformaldehyde prior to acquisition. Live cells were gated based on forward and side scatter.
For intracellular staining of γH2AX, surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 °C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing 3.3 µg/mL 7-AAD.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).
For intracellular staining of γH2AX, surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 °C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing 3.3 µg/mL 7-AAD.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).