CAR T cells were stained with a Live/Dead Fixable Near-IR stain (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, followed by a phosphate-buffered saline (PBS) wash and incubation with a ChromPure Human IgG blocking solution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 5 min. An antibody cocktail containing anti-PSMA antibody (clone LNI-17; BioLegend, San Diego, CA, USA), anti-EGFRt (Erbitux; Lilly, Indianapolis, IN, USA), anti-CD4 (clone OKT4; BioLegend), anti-CD8 (clone RPA-T8; BioLegend), and an in-house–generated anti-idiotypic antibody to detect the CAR was added for 20 min. Cells were then washed twice with staining buffer (BioLegend) and analyzed on a NovoCyte cytometer (ACEA Biosciences, San Diego, CA, USA). Data analysis was performed using FlowJo v10.1 software (Tree Star, Ashland, OR, USA).

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