Differential response of eWAT macrophage subpopulations to systemic acute inflammatory challenges. (A–F) WT mice were injected with 50 µg of LPS i.v., and the eWAT myeloid cell composition was determined 12, 24, and 120 h after injection. (B) t-SNE map displaying the eWAT CD45⁺ cell landscape from untreated or LPS-treated WT mice, analyzed by flow cytometry. Colors correspond to Cytobank-guided clustering of cell populations. (C) Flow cytometry. (D) Absolute number of eWAT myeloid cells. (E) Epididymal adipose tissue weight. (F) SVF and total CD45⁺ absolute cell number. Representative data of three independent experiments (n = 3–5 mice per experiment). (G and H) Functional fate mapping of VAMs exposed in vivo to LPS. WT mice were injected i.v. with dextran-rhodamine 70 kD. 36 h later, the same animals were injected i.v. with 50 µg of LPS. 24 h after the LPS injection, eWAT macrophages were analyzed by flow cytometry. Data representative of two independent experiments (n = 3 per group). (I and J) eWAT myeloid cell proliferation determined by BrdU incorporation at different time points. WT animals were injected i.p. twice with 1 mg of BrdU, 12 h apart. BrdU injections started concomitantly with the LPS injection or 48 h after LPS injection. Animals were analyzed at 24- or 72-h time points. Representative data from two independent experiments (n = 3–5 per group). (K–O) WT mice were inject i.v. with 10⁸ CFU of S. enterica serovar Typhimurium (Salmonella) and eWAT myeloid cell composition was determined 3 and 6 h after infection. (L) Flow cytometry. (M) Epididymal fat weight. (N and O) SVF, total CD45⁺, and eWAT myeloid cell population, absolute numbers. Representative data of two independent experiments (n = 3–5 mice). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Statistical analysis was performed using one way ANOVA together with Tukey post hoc test. AT, adipose tissue. Animals were 12–15 wk old. Bar graphs are shown as mean ± SEM.