FIGURE 5.

Gating strategy for lung cell phenotyping. The fully stained sample was unmixed with AF included in the ID7000 software, then FCS files were exported and opened in FlowJo. Sequential gating was used to identify cell populations. Data clean-up included time gating (A), doublet exclusion (B), viability gating (C), and debris removal (D). CD45 and Sca-1 were used to discriminate 3 populations (E): hematopoietic cells (CD45⁺; red gate), endothelial cells (ECs) (CD45⁻/Sca-1⁺; pink gate), and non-hematopoietic/non-EC cells (CD45⁻/Sca-1⁻; yellow gate). A scatter plot (G) and expression of C-kit and Sca-1 defined proangiogenic cells (PACs) (H). CD11c⁺/CD11b⁻ cells were gated (I) then expression of Siglec-F and absence of CD24 was used to gate alveolar macrophages (J). CD11b⁺ cells (K) were gated from the remaining cells in plot I. CD24 was expressed on granulocytes (L) then Siglec-F⁺ eosinophils and Ly-6G⁺ neutrophils were gated (M). The remaining cells in plot L were selected and MHC II⁺ cells were gated (N). CD64⁺/CD24⁻ cells were gated as interstitial macrophages (O). From the EC gate (pink gate in plot (P)], CD90.2 and VWF were used to gate pulmonary EC subsets as alveolar ECs (CD90.2-/VWF-), lymphatic ECs (CD90.2+/VWF-), or blood vessel ECs (CD90.2-/VWF+) (R). From the non-hematopoietic/non-EC subset (yellow gate in plot (Q), α-SMA⁺ cells were gated (U) then myofibroblasts expressing low or high amounts of collagen-1 were gated (V). Also from the non-hematopoietic/non-EC subset, PDGFRb⁺/CD146⁺ pericytes (S), and MUC2⁺/EPCAM⁺ epithelial goblet cells (T) were gated.