Multiparametric Flow Cytometry Assay

Fluorochrome-conjugated antibodies against CD3, CD8, CD44, CD62L, CD69, Tim-3, PD-1, CD90.1, CD45.1, Granzyme B, Tbet, Bcl-2, IFN-γ, IL-2 (Biolegend), NKG2D, CD25, TNF-α, KLRG1 (eBioscience), pSTAT5 (Invitrogen), CD127 (BD Bioscience) and pS6 (Cell Signaling Technologies) were used. Cell surface staining, intracellular staining, and flow cytometry analysis was performed as previously described [28 (link)]. Staining of phosphorylated proteins was performed following eBioscience protocol with methanol fixation and permeabilization. For analyses, cells were gated on live cells using Zombie Aqua exclusion dye (Biolegend). pMel cells were distinguished from endogenous cells by gating on the congenic marker CD90.1 and in vivo CTL assay analyses were performed after gating on the congenic marker CD45.1. When methanol was used, live cells were defined based on size.