Figure 1. Isolation of Lin⁻LEPR⁺ and Lin⁻SCA1⁺ BM-MSCs.

a, Schematic showing timeline and enrichment method used to generate S, ID, and Ex BM-MSCs. BM was pooled from n=3–5 mice per isolation. b, Representative dot plots of flow cytometric data showing gating strategy used to isolate the Lin⁻LEPR⁺ fraction from BM following red cell lysis; forward vs. side scatter (left, top), single cell gating (right, top), viability staining with PI (left, bottom), and final sort gate (right, bottom). c, Representative dot plot showing gating of Lin⁻LEPR⁺ cells after pre-depletion with anti-CD45, anti-CD11b, and anti-CD34 antibodies. d, Table of Lin⁻LEPR⁺ cell yields following isolation by RBC lysis vs. preimmuno-depletio n. e, Represe ntative dot plots of flow cytometr y data showing BM cells stained with anti-CD44 and anti-CD45 antibodi es pre (left panel) and post immuno-depletion (right panel). Bar graph shows fold enrichment of Lin⁻SCA1⁺ ID BM-MSCs based on CD44 and CD45 staining. Data are mean ± SD from three biological replicates. f, Representative dot plots of flow cytometry data showing surface phenotype characterization of Ex BM-MSCs.