Isolation and identification of BM and endosteal nonhematopoietic niche cells. (A) Scheme of niche cell isolation. BM and bone pieces are treated with collagenase type I to disintegrate tissues. (B-C) Identification of nonhematopoietic niche cells in the marrow fraction (B) and in the endosteum (C) by flow cytometry. Samples were gated on size, singlets, and viable cells (Dapi⁻). Lin = CD3, CD4, CD8, Gr1, CD11b. (D-E) Frequency (left; n = 47; 29 experiments) and absolute numbers (right; n = 33; 21 experiments) of marrow (D) and endosteal (E) niche cells in C57BL/6J wild-type mice. Absolute numbers for BM: Endo = 2506 ± 2315; MSC = 11 811 ± 3956; OP = 7791 ± 9386; PαS = 113 ± 141. Absolute numbers for bone: Endo = 1867 ± 1688; MSC = 3951 ± 3206; OP = 1944 ± 1419; PαS = 1086 ± 1249. (F) Distribution of niche cell types between the marrow fraction (shaded) and endosteum (bright colors). Calculations are based on absolute cell numbers; n = 50, 30 experiments. Endo, endothelial cells.