Zombie yellow viability dye

Unconjugated humanized anti‐GM2 mAb (Creative Biolabs) and secondary APC‐conjugated anti‐human IgG Fc mAb (clone HP6017; BioLegend) were used to detect the surface GM2. The CAR‐expressing T cells were detected with APC‐Cy7‐conjugated anti‐CD3 mAb (clone HIT3a; BioLegend), PerCPCy5.5‐conjugated anti‐CD4 mAb (clone OKT4; BioLegend), APC‐conjugated anti‐human CD8 (clone HIT8a; BioLegend), and/or biotinylated anti‐idiotype Ab against anti‐GM2 CAR (Cell Engineering Corporation), followed by PE‐conjugated streptavidin (BioLegend). Zombie Yellow viability dye (BioLegend) and PE‐conjugated anti‐CD45 mAb (clone HI30; BioLegend) were used to detect viable immune and non‐immune cells after in vitro co‐culture assay. Human TruStain FcX (BioLegend) was used to block nonspecific binding of mAb with Fcγ receptors. Flow cytometric data were acquired and analyzed as previously reported.¹⁰

For FACS sorting of single cells, BM cells were isolated and RBC lysed as described above. This was followed by lineage depletion using a lineage antibody cocktail against CD4, CD8, CD11b, B220, Gr-1, and Ter119 and incubation with Dynabeads Magnetic Beads (Invitrogen). Lineage-depleted BM cells were stained with Zombie Yellow viability dye (BioLegend) followed by incubation with the following antibodies: CD117, Sca1, CD150, CD48, CD34, and lineage antibodies (B220, CD4, CD8, Ter119, Ly6G, CD11b, CD11c, and F4/80) together with one of the hash antibodies (TotalSeq-A0301 anti-mouse Hashtag 1 Antibody, TotalSeq-A0302 anti-mouse Hashtag 2 Antibody, TotalSeq-A0303 anti-mouse Hashtag 3 Antibody, TotalSeq-A0304 anti-mouse Hashtag 4 Antibody) (TotalseqA antibodies; BioLegend). The four biological replicates of each time point were stained with one of the four unique hash antibodies. Cells were sorted using a FACSAria Fusion or FACSAria II equipped with a 100 μm nozzle (BD Biosciences).