Cytotoxicity Assay by Flow Cytometry.

38c13 target cells labeled with PKH67 according to the manufacturer’s procedure (Sigma-Aldrich) were cocultured at a ratio 1:10 with sCAR and conventional CAR T cells (1 × 10⁴ targets with 1 × 10⁵ effectors) previously CAR⁺ enriched and normalized (to a 50 ± 6% transduction efficiency with nontransduced cells; SI Appendix, Fig. S1B) and 1 nM anti-mouse CD19 LCNT switch for 6 h at 37 °C. Supernatants were frozen for further analysis, and cells were stained with the Zombie Red Fixable Viability kit (BioLegend) and fixed with 2% paraformaldehyde final (Electron Microscopy Sciences). Five microliters of CountBright Absolute Counting Beads (Molecular Probes) were added to the cells. A fixed volume of samples was acquired on an LSRFortessa X-20 flow cytometer (BD Biosciences) and analyzed by using FlowJo software (version 10.1). The number of viable target cells remaining (Zombie Red⁻/PKH67⁺) and beads were used to calculate the number of cells per microliter following the CountBright manufacturer’s instructions. The cytotoxicity against the target cells (percentage target killed) was calculated as 100 × [(number of live target cells per microliter in target + effector cells, no switch) − (number of live target cells per microliter in target + effector cells, switch)/(number of live target cells per microliter in target + effector cells, no switch)]. Cytokines in the supernatants were quantified by using Cytometric Bead Array (CBA) Mouse Soluble Protein Flex Set kits (BD Biosciences) according to product manuals after acquisition on an Accuri C6 flow cytometer (BD Biosciences) and analysis with FCAP Array software (BD Biosciences).