Immunofluorescence Staining of hBMMSCs

The third-passage hBMMSCs at 50%–70% confluence were digested with 0.25% trypsin-EDTA and prepared into single-cell suspension. After the cell concentration was adjusted to 2 × 10⁵/mL, they were seeded into a 6-well plate, and incubated in a 5% CO₂/37°C incubator overnight. On the next day, immunofluorescence staining was performed as follows: (1) The cells were rinsed with PBS 3 times, fixed with 4% paraformaldehyde for 30 min at room temperature, and rinsed again with PBS 3 times. (2) They were blocked with PBS containing 1% BSA for 1 h, and rinsed with PBS 3 times. (3) Primary antibodies (mouse anti-human CD34 hematopoietic stem cell surface molecule or mouse anti-human CD44 MSC surface molecule) were added for incubation at 37°C for 2 h. (4) Fluorescent secondary antibodies were added for incubation at 37°C for 60 min in the dark, the cells were washed with PBS 3 times, and residual PBS was sucked dry using a pipette tip. (5) Hoechst (1 mg/mL; Yeasen Biotechnology (Shanghai) Co., Ltd., China) was added for nuclear staining for 15 min in the dark, and the final Hoechst concentration was 1 μg/mL. The expression of cell surface markers was observed under a fluorescence microscope.13 (link)