Determination of protein copy number per cell

The average antigen density per target cell was determined by quantitative flow cytometry. For each of the target antigens or T cell receptors, 1x10⁵ cells of each population were stained with the desired antibody: Anti-HER2 APC (Biolegend 324407), Anti-EGFR BV786 (BD Biosciences 742606) or Anti-myc Alexa 647 (CellSignaling 2233S) antibody for 30 min on ice (n=3) in 96-well plates. Cells were washed twice with PBS (200 uL/well) and resuspended in PBS (100 uL) for analysis in an Attune NxT Flow Cytometer. The geometric mean of each target population was determined after gating the cells by their size (side scatter and forward scatter region) and selecting the full width at half maximum (FWHM) of the population in the corresponding fluorescent channel. A standard curve was built using Quantum Simply Cellular anti-Mouse IgG beads (Bang Laboratories 815) stained with the same antibody used for the target cells. For each cell line, the number of molecules per cell was determined using the standard curve and the geometric mean of each target population. Similarly, the expression amounts of inducible CAR expression were determined using mCherry flow cytometer calibration beads (Takara Bio 632595).