Evaluating Immune Cell Signaling Pathways

For all signaling assays, non-sorted splenocytes or lymph node cells were serum-starved for 1 h at 37 °C before stimulation in complete media without serum. For evaluation of TCR signaling, splenocytes and lymphocytes were stimulated with 20 μg/mL anti-CD3ε (145-2C11; Biolegend) and 50 µg/mL anti-IgG (polyclonal, α-Armenian hamster, Jackson ImmunoResearch) for the indicated times^{69 (link),70 (link)}. For evaluation of pSTAT5 signaling, cells were stimulated for 15 min with recombinant human IL-2 (rhIL-2; NIH AIDS Reagent Program) at indicated concentrations, recombinant mouse IL-15 (rmIL-15; Peprotech) at indicated concentrations, or 10 ng/mL recombinant IL-7 (rmIL-7; Peprotech). Immediately following stimulation cells were fixed with a final concentration of 1.5% methanol-free formaldehyde (Fisher Scientific; Cat. PI28906) for 15 min, and permeabilized with 500 µL of ice-cold methanol per 1 × 10⁶ cells for 30 min^{69 (link),71 (link)}. Cells were washed twice with PBS/2%FBS (Omega Scientific) then stained for 60 min in anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-FoxP3 (FJK-16s), anti-pSTAT5 (Y694; C71E5; Cell Signaling Technologies) or anti-pS6 (S235/236; D57.2.2E; Cell Signaling Technologies), and, for exclusion of non-relevant cells, anti-CD45R (B220; RA3-6B2), anti-CD11b (MI/70), anti-CD11c (N418), and anti-Ly-6G (Gr-1; RB6-8C5).