4 6 diamidino 2 phenylindole dihydrochloride dapi

To confirm cytotoxic effects on a single cell level in pilot experiments, human HaCaT keratinocytes were harvested in FACS tubes using accutase. Cells were washed three times with cold FACS washing buffer (Miltenyi Biotec, Mönchengladbach, Germany), and cellular viability was determined by adding 1 µM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; BioLegend, Amsterdam, The Netherlands). Flow cytometry experiments were performed using a CytoFLEX LX device (Beckman-Coulter, Krefeld, Germany), and data were analyzed using Kaluza 2.1 software (Beckman-Coulter, Krefeld, Germany).

Before staining with relevant fluorochrome-conjugated Abs, cells were treated with Human TruStain F_cX (F_c receptor blocking solution; BioLegend, San Diego, CA, USA, #422302). Abs against CD4 (APC; OKT4; #317416), CD8 (PerCP-Cy5.5; HIT8a; #300924), TIGIT (PE-Cy7; A15153G; #372713), CD226 (FITC; 11A8; #338303), CD155 (Alexa Fluor 647; SKII.4; #337621), CD111 (PE; R1.302; #340404), HER2 (PE; 24D2; #324405) and mouse IgG₁ κ isotype control (PE; MOPC-21; #400113) were from BioLegend, San Diego, CA, USA. Ab against CD96 (PE; NK92.39) was from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA (#12-0969-42) or BioLegend, San Diego, CA, USA (#338405). Cell surface expression of CAR was detected by incubation with recombinant biotinylated protein L (ACROBiosystems, Newark, DE, USA, #RPL-P814R) followed by incubation with PE-conjugated streptavidin (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA, #12-4317-87). To exclude dead cells, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Biolegend, San Diego, CA, USA, #422801) was used. Samples (at least 20,000 events) were acquired on a LSRII cytometer (BD Biosciences) or MACSQuant X (Miltenyi Biotec) and analyzed with FlowJo software (TreeStar, BD Life Sciences, Franklin Lakes, NJ, USA).