Flow cytometry gating strategies. (a) For mouse characterization studies (Fig. 1), cell suspensions prepared from the lymphoid organs or blood were gated on single cells and live cells, then the indicated lineage-defining surface markers. (b) Gating on naive CD4⁺ T cells to determine surface S1PR1 expression, corresponds to Fig. 4 a. (c) Gating showing the mixed population of CellTracker Green–labeled WT (CD45.1⁺) and DKO (CD45.2⁺) T cells. Input refers to what was injected into recipient animals; harvest refers to lymphoid organs and blood harvested 1 or 24 h later. Corresponds to Fig. 2, g and h. (d) Gating on singlets and CD4⁺CD8⁻ T cells for actin polymerization studies. Note that the fixation strategy used in these studies precludes live/dead staining. (e) Gating on singlet CD4⁺ cells for in vitro S1PR1 endocytosis studies. Small amounts of extremely high S1PR1⁺ cells that skewed quantitation and were later confirmed to be dead cells were gated out. FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W, forward scatter width; SSC-W, side scatter width.