Cytotell red 650

Antibodies used in this study: anti-hamster IgG, whole molecule (Sigma-Aldrich, St. Louis, MO), NA/LE hamster anti-mouse CD3ε, anti-mouse CD28 (BD Biosciences), anti-PRMT5 (A-11, Santa Cruz Biotechnology, Dallas, Tx), anti-H3R2me2s polyclonal (Invitrogen, Waltham, MA), alpha-tubulin mouse (DM1A, Cell Signaling Technologies (CST), Danvers, MA), anti-mouse secondary-IgG HRP linked F(ab’)₂ fragment (clone NA9310V Amersham Biosciences, Piscataway, NJ), anti-rabbit secondary – IgG HRP linked whole antibody (clone NA934V, Amersham Biosciences), anti-PRMT5 rabbit monoclonal (clone ST51-06, Invitrogen), anti-H3R2me2s ChIP-seq grade (EpigenTek, Farmingdale, NY), anti-CD4 FITC (Clone H129.19, BD Biosciences), anti-CD25 (clone PC61, BioLegend, San Diego, CA), anti-Foxp3 (clone MF-14, BioLegend), anti-IFNγ (BD Biosciences), anti-Tbet (BioLegend), nuclear stain Draq5 (ThermoFisher Scientific), anti-rabbit IgG Fab₂ Alexafluor 488 (CST), zombie violet fixable viability kit (BioLegend), CytoTell UltraGreen and CytoTell Red650 (AAT Bioquest, Pleasanton, CA).

We examined the proliferation of CAR-T-cells using CytoTell™ Red 650 (AAT Bioquest Inc) dye dilution after serial stimulation with tumor cells. Briefly, CAR-T cells were incubated with CytoTell™ Red 650 dye at 37°C for 30 min, and the dye working solution was removed. Then, the CAR-T cells were co-cultured with tumor cells at a ratio of 1:1 or without tumor cells (no stimulation). After 3 days of co-culture, the proliferation of CAR-T cells was analyzed by flow cytometry, and the experiments were repeated for serial three rounds of antigen stimulation.