Extended Data Fig. 2: Purification of smooth muscle and fibroblast cells from aorta (Related to Fig. 3).
From: Defining the lineage of thermogenic perivascular adipose tissue
a, FACS isolation of fibroblastic and smooth muscle cell (SMC) populations. Dissociated cells were gated on: (1) SSC-A and FSC-A to exclude debris; (2) FSC-H vs. FSC-W then SSC-H vs. SSC-W to isolate single cells; and (3) live (FVS510-) Lin- (CD45-,CD31-,Ter119-) cells. Depicted sort gates were used to isolate the following populations: Progenitors [LY6A high], Preadipocytes (PreAd) [LY6A(-), CD142 mid; CD200(-)]; SMCs [LY6A(-); CD142(-); CD200+], Intermediate Cells (Int) [LY6A(-); CD142+; CD200+, CD317(-)], Meso (Mesothelium) [LY6A(-); CD142+; CD200+, CD317+] (Representative images from n=10 expts). Related to experiments shown in Figs. 3a and 5. b, Violin plots showing expression of genes used in the sorting strategy. c, Staining of CD200 (red), MYH11 (green), and DAPI (blue) in P3 thoracic aorta. White arrowhead shows an Intermediate cell. Yellow arrowhead shows an SMC (scale bar, 65 μm; Lu: lumen). Representative of n=1 experiment. (d) Expression of Seurat cluster-defining genes in the sorted cell bulk RNAseq datasets. Gene levels in sorted cell populations were compared to the row mean and annotated as “enriched” or “de-enriched” (Log2 FC>0.25).
