Human cells were stained with the following fluorochrome-labeled antibodies: anti-CD45-PECy7 (Biolegend), anti-CD8-BV510 (Biolegend), anti-CD25-BV421 (Biolegend), anti-CD69-PerCPCy5.5 (Biolegend), anti-CD137-Biot (5D1, the laboratory’s own hybridoma), anti-Ki67-AF488 (Biolegend) and anti-Granzyme-AF647 (Biolegend). Streptavidin-PE was added to detect 5D1-Biot Ab. Isotype control mixes was prepared with mIgG1-BV421 (Biolegend), mIgG1-PerCPCy5.5 (Biolegend), mIgG1-AF488 (Biolegend), mIgG1-AF647 (Biolegend). In the case of 5D1-Biot, FMO was performed. M11 CAR expression was verified with anti-mIgG(H+L)-AF647 (Invitrogen) just before stimulation with mesothelin-Fc-beads.
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).
Mouse T cells were stained with: anti-CD8-PECy7 (Biolegend), anti-CD25-APC (Biolegend), anti-CD69-BV510 (Biolegend), anti-CD137-PE (Biolegend), anti-PD1-PerCPCy5.5 (Biolegend), and anti-Ki67-AF488 (BD Bioscience). Rat IgG1-APC (Biolegend), Arm Hamster-BV510 (Biolegend), Syr Hamster-PE (Biolegend), Rat IgG2a-PerCPCy5.5 (Biolegend) and mIgG1-AF488 (Biolegend) antibodies were used as isotype-matched negative controls.
Zombie NiR (Biolegend) was used to exclude cell death. Samples were acquired on a BD Canto II (BD Biosciences) and CytoFlex S systems (Beckman Coulter). Analyses were performed using FlowJo (Tree Star) and CytExpert software (Beckman Coulter).