Cd105 pe

The cells were cultured in high glucose α-MEM (Gibco, USA), 10% FBS (Gibco, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin for 1 week, culture medium was replaced twice a week. Cells were collected and counted, then re-suspended with α-MEM, we adjusted the cell density at 1 × 10⁶ cells/ml. Each 0.1 ml sample was incubated with 20 μl of CD73-PE, CD90-FITC, CD34-PE, CD45-PE, CD105-PE (Santa Cruz, USA) at 4 °C for 1 h. Antibody binding was analyzed by flow cytometry (Becton Dickson, USA).

mADSCs and rADSCs at P3 were harvested by enzymatic digestion, centrifuged and washed with PBS. The cells were then stained with anti‐mouse antibodies against CD34‐APC (Biorbyt, Cambridge, Cambridgeshire, UK), CD45‐APC (BioLegend, San Diego, CA, USA), CD90‐APC (BioLegend) and SCA‐1‐APC (BioLegend) or anti‐rat antibodies against CD34‐PE (BioLegend), CD45‐PE (BioLegend), CD90‐PE (BioLegend) and CD105‐PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA). mADSCs and rADSCs at passage 1 (P1)‐ passage 6 (P6) or isolated CD49f⁺ rADSCs were stained with anti‐CD49f antibody (mADSCs: Invitrogen, ThermoFisher Scientific, Waltham, MA, USA; rADSCs: Bio‐Rad). To investigate whether the expression of CD49f in ADSCs was influenced by inflammatory environment, ADSCs at P2 were treated with 40 ng/mL TNF‐α (Sigma‐Aldrich) or 20 ng/mL IFN‐γ (R&D Systems) for 24 hours and then stained with anti‐CD49f antibody (mADSCs: Invitrogen, ThermoFisher Scientific; rADSCs: Bio‐Rad) and anti‐VCAM‐1 antibody (mADSCs: BioLegend; rADSCs: BioLegend).
The cells were kept in the dark for 30 minutes and then washed twice with PBS. Cytometric analysis was performed using a BD FACSCelesta™ flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and the results were analysed using FlowJo software.