Anti cd29 fitc

Approximately 1 × 10⁶ PDLSCs at the third passage were added in blocking buffer and incubated with anti-STRO-1 immunoglobulin (Ig)M (Santa Cruz, CA, USA) for 1 h at 37°C, then incubated with goat anti-mouse IgM FITC secondary antibodies (Molecular Probes, Eugene, Carlsbad, CA, USA) for an additional 1 h. PDLSCs incubated with the secondary antibody only was used as a negative control. Another 1 × 10⁶ PDLSC was incubated with anti-CD146-PE, anti-CD45-FITC, and anti-CD90-APC antibodies (BD Biosciences, USA), at 37°C for 30 min. 1 × 10⁶ BMSCs at the third passage were incubated with antibodies at 37°C for 30 min, including anti-CD29-FITC, anti-CD 45-FITC, anti-CD44-FITC, anti-CD11b/c-FITC, and anti-CD90-FITC (BioLegend, CA, USA). The cells were then analyzed by flow cytometry (Attune NxT, Invitrogen, USA) and Treestar FlowJo software.

The mice were subjected to cervical dislocation following anesthesia with isoflurane. The mice were then sterilized with 70% ethanol for 5 min. Mouse BMMSC were isolated from the femurs and tibias of wild‐type or KAT2A^+/− C57BL/6 mice (male, 6–8 weeks, 20–30 g). The femurs and tibias were dissected and cleaned of connective tissue. Both ends of the femurs and tibias were incised, and the bone marrow cells were washed out with PBS, and filtered through a 70‐µm cell strainer. The collected cells were centrifuged at 1500 rpm for 5 min, resuspended in a complete MesenCult expansion medium (STEMCELL, # 05513) containing 10% FBS, 100 IU mL⁻¹ penicillin, and 100 IU mL⁻¹ streptomycin, and seeded into 25 cm² flasks and cultured at 37°C in an atmosphere with 5% CO₂. The nonadherent cells were removed after 3 days, and half of the medium was refreshed every 3 days. At 90% confluence, the cells were passaged using 0.05% trypsin. The phenotypes of the expanded mouse BMMSC were detected by flow cytometry analysis. BMMSC were harvested, washed with PBS, and incubated with anti‐CD45‐PE (BioLegend, #103105), anti‐CD11b‐FITC (BioLegend, #101205), anti‐CD34‐PE (BioLegend, #119307), anti‐CD29‐FITC (BioLegend, #102205), anti‐CD105‐FITC (Invitrogen, #MA5‐17945), anti‐Sca1‐PE (BioLegend, #108107) and anti‐CD44‐PE (BioLegend, #103023), and analyzed by flow cytometry.

P3 BMMSCs were digested with trypsin and resuspended in DMEM containing 10% FBS. The cells were washed with phosphate-buffered saline (PBS) twice. Subsequently, the cells were incubated with anti-CD11b/c-PE (#554862, BD Pharmingen, USA), anti-CD34-PE (#sc-7324, Santa Cruz Biotechnology, USA), anti-CD29-FITC (#102205, Biolegend, USA), and anti-CD90-FITC (#561973, BD Pharmingen, USA) antibodies for 30 minutes. The cell suspension was then centrifuged at 1000 rpm for 5 minutes. Finally, the cell suspension was transferred into a new detection tube, followed by the detection of cell surface antigen using flow cytometry (BD Biosciences, San Jose, CA, USA).

The Epi‐SCs were analyzed through flow cytometry, as described previously.³³ Briefly, the cells were suspended in PBS containing 1% bovine serum albumin at a density of 10⁶ cells/ml. Cell aliquots (100 μl) were incubated with different fluorescence‐conjugated monoclonal antibodies, namely anti‐CD29‐FITC (1:25, 102205, Biolegend) and anti‐CD49f‐PE (1:25, 313611, Biolegend), or the control isotype IgG at 4°C for 30 min. Flow cytometry (CytoFLEX, Beckman) was used to analyze 10,000 events using Cell Quest software.