Flow cytometric and single-cell transcriptomic analysis of FRCs and VSMCs in peripheral and mesenteric lymph nodes. (A–D) Representative flow cytometric analysis of peripheral (A and B) and mesenteric (C and D) lymph nodes of 8-wk-old Ccl19-iEYFP mice. Manual gating strategy used for gating of FRC and VSMC populations in peripheral (A) and mesenteric (C) lymph nodes. Relative expression of the indicated surface markers projected on the UMAP representation of FRC and VSMC populations of peripheral (B) and mesenteric (D) lymph nodes. Data are representative for n = 15 mice from four independent experiments (A and C) and n = 11 mice from three independent experiments (B and D). (E and F) Quantification of Cxcl13-EYFP⁺ cells gated according to the gating strategy shown in Fig. S2, A and C with pre-gating on CD31⁻ cells. Data are shown as the mean and SEM from n = 7 (E) and n = 6 mice (F) from two independent experiments. (G) Schematic representation of the experimental approach used for the single-cell transcriptomics analysis. (H) Representative sorting strategy of non-hematopoietic cells isolated from peripheral and mesenteric lymph nodes of Ccl19-iEYFP mice. (I) Representative sorting strategy of non-hematopoietic cells isolated from peripheral and mesenteric lymph nodes of Cxcl13-EYFP mice. (J) Dot plot showing the average expression of signature genes in FRC subsets and VSMCs isolated from Ccl19-iEYFP mice compared between peripheral and mesenteric lymph nodes. (K) Top 15 differentially expressed genes upregulated in FRC subsets and VSMCs from peripheral and mesenteric lymph nodes. Lymph node scRNA-seq data from Ccl19-iEYFP is representative of n = 15 mice from four independent experiments. Figure was supplemented with elements from https://BioRender.com.