eWAT harbors a complex distribution of immune cells containing four macrophage subpopulations. (A) Abbreviated description of the key surface markers defining the cell populations analyzed. The colors identifying each population are used throughout the manuscript. (B) t-SNE map displaying the distribution of 200,000 CD45⁺ cells from the eWAT of steady state WT mice, analyzed by flow cytometry. Colors correspond to Cytobank-guided clustering of cell populations. Representative plot of three independent experiments (n = 3). (C) t-SNE map displaying the distribution of 100,000 CD45⁺CD11b⁺ CD90⁻CD19⁻Ly6G⁻SiglecF⁻ cells from the eWAT of steady state WT mice analyzed by flow cytometry. Colors correspond to Cytobank-guided clustering of cell populations. Representative plot of three independent experiments (n = 3). (D) PCA of the transcriptional signature obtained by RNAseq data from the populations described in A. Adult (average 16-wk-old) WT males were used. Each dot represents data from a pool of two to three epididymal fat pads. (E) Unbiased gene expression profile of eWAT myeloid subpopulations described in A, showing all genes for broad visualization. Each column represents consolidated data from three biological replicates per cell type. The z-score of the gene expression profiles gives a scale to measure the differential expression. (F) Heat map of transcription factors differentially expressed among eWAT myeloid subpopulation. The yellow boxes depict clusters of transcription factors predominantly expressed by DCs (DC cluster), monocytes (eMon cluster), and VAMs (VAM cluster) in the eWAT in homeostasis. Each column represents consolidated data from three biological replicates. The z-score of the gene expression profiles gives a scale to measure the differential expression. Adult (average 16-wk-old, range 12–20) WT males were used.