Figure 1. Gating strategy for defining major subsets of leukocytes among splenocytes. A.

Dead cells were removed from the analysis using Live/Dead® fixable dead cell stain. B. Doublets were removed from living cells (Live/Dead⁻) using FSC-A and FSC-H. C. Gr-1^bright splenocytes were gated and defined as neutrophils. D. NKp46⁺ events were gated from Gr-1^low/− splenocytes and defined as natural killer (NK) cells. E. NKp46⁻, Gr-1^low/− splenocytes were divided into MHC II⁺ and MHC II⁻ populations. F. MHC II⁺ events were split by B220 and CD11c expression. MHC II⁺, B220⁺, CD11c⁻ events were defined as B cells. MHC II⁺ B220⁻, CD11c⁺ events were defined as dendritic cells (DCs). G. We split MHC II⁻ events by CD3 and CD11b expression. CD11b^-, CD3⁺, MHC II^- events were labeled as T cells. H. CD11b⁺, CD3⁻, MHC II^- events were split according to SSC profile. Events with higher SSC values (suggesting greater internal granularity) were labeled as eosinophils. I. Events with lower SSC values were divided by Gr-1 expression, giving two populations: Gr-1(Ly-6C)^high monocytes/macrophages and Gr-1(Ly-6C)^low monocytes/macrophages (mono/MΦ).