After 2-week culture in α-MEM plus 10% FBS, mouse BMSCs were trypsinized, pelleted and then resuspended in PBS containing 2% FBS to a concentration of 2 × 106 cells/ml. Freshly isolated bone marrow cells were resuspended to a concentration no more than 1 × 107 cells/ml. Cells were stained with anti-mouse CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), anti-mouse/rat CD29 PE-Cy7 (eBioscience, 25-0291-80), anti-mouse CD105 (Endoglin) PE (eBioscience, 12-1051-81), and anti-mouse Ly-6A/E (Sca-1) APC (eBioscience, 17-5981-81), and analyzed with an LSRII cytometer (Becton Dickinson, San Jose, CA, USA). For BrdU Incorporation, the mice were intraperitoneally labeled twice (a 16-h interval) with 1 mg/mouse/injection BrdU and euthanized 2 h after the second injection. Freshly harvested bone marrow was treated with 1× RBC Lysis Buffer (eBiosciences, San Diego, CA, USA). The remaining cells were stained with anti-mouse CD45 PerCP-Cy5.5, anti-mouse/rat CD29 PE-Cy7, anti-mouse CD105 (Endoglin) PE, and anti-mouse Ly-6A/E (Sca-1) Alexa Fluor 700 (eBioscience, 56-5981-82) before permeabilization, DNase treatment and staining with anti-BrdU APC according to the instruction of APC BrdU Flow Kits (BD Pharmagen, Franklin Lakes, NJ, USA). The percentage of BrdU-positive fractions was assessed using FlowJo analysis software (Tree Star, Ashland, OR, USA).
Huang J., Zhao L., Fan Y., Liao L., Ma P.X., Xiao G, & Chen D. (2019). The microRNAs miR-204 and miR-211 maintain joint homeostasis and protect against osteoarthritis progression. Nature Communications, 10, 2876.
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