(A) Immunofluorescent staining of CD31^–CD45^–Sca-1⁺ cells in mouse bone marrow of WT and Nell-1^+/6R mice, analyzed at 3 months of age. From left to right: CD31/CD45⁺ cells appear green, stem cell antigen 1⁺ (Sca-1⁺) cells appear red, CD31/CD45⁺Sca-1⁺ cells appear yellow, and on the far right CD31^–CD45^–Sca-1⁺ cells appear red after digitally removing all yellow fluorescence. Scale bars: 50 μm. (B) At high magnification, the localization and type of CD31^–CD45^–Sca-1⁺ cells (arrows) was revealed, with the majority of cells residing in an intramarrow location, with scattered bone lining cells stained. Dashed white lines indicate margins of trabecular bone. DAPI nuclear counterstain appears blue. Scale bars: 25 μm. (C) Semiquantification of staining presented in A. Six random fields were analyzed per slide with 3 slides analyzed per sample. (D) Representative flow cytometry methods for Sca-1⁺ cell quantification. After selecting for the CD31^–CD45^– population (P2), and positive selection for the mesenchymal progenitor cell (MPC) markers CD105 and CD44 (P3), the subpopulation of Sca-1⁺ (P4) was then selected. (E) Flow cytometric analysis of CD31^–CD45^–CD105⁺CD44⁺Sca-1⁺ population in mouse bone marrow of WT and Nell-1^+/6R mice, analyzed at 3, 7, and 18 months of age. Percentage of cells expressed as CD31^–CD45^–CD105⁺CD44⁺Sca-1⁺ cells over total CD31^–CD45^– cells. (F) Flow cytometric analysis of the CD31^–CD45^–CD105⁺CD44⁺Sca-1⁺ population in mouse bone marrow of CMV-Cre⁺Nell-1^+/+ and CMV-Cre⁺Nell-1^fl/fl mice at 3 months of age. Percentage of cells expressed as CD31^–CD45^–CD105⁺CD44⁺Sca-1⁺ cells over total CD31^–CD45^– cells. Data reported as mean ± SEM. *P < 0.05 and **P < 0.01 compared with respective controls. Analyzed using a 2-tailed Student’s t test (C and F) or a 1-way ANOVA followed by a post-hoc Tukey’s test to compare between 2 groups (E).