Anti cd200

Flow cytometry was performed on a FACS Beckman analyzer. All data were analyzed using FlowJo software. Digested chondrogenic cells were re-suspended in FACS buffer (2%FBS in PBS) and stained with anti-CD45 (1:1000, eBioscience), anti-CD31 (1:1000, eBioscience), anti-Ter119 (1:1000, eBioscience), anti-CD90.2 (1:500, Biolegend, 105335), anti-CD51 (1:100, eBioscience, 13-0512-82), anti-CD200 (1:100, eBioscience, MA5-17980), anti-CD105 (1:100, eBioscience,17-1051-80) primary antibody for 1 h at 4 °C, following by Streptavidin, Pacific Blue™ conjugate (1:200, Thermo, S11222) for 30 min at 4 °C. After PBS wash, cells were analyzed by flow cytometry for Gli1⁺ (CD45⁻CD31⁻Ter119⁻tdTomato⁺) and Gli1⁻ (CD45⁻CD31⁻Ter119⁻tdTomato⁻) cells. Data were analyzed with FlowJo version 10.

Single-cell suspensions were prepared from the cultured cells as described above. The cells were rinsed once with PBS and incubated with anti-ITGα6 (1:200, eBioscience, San Diego, CA, United States) and anti-CD200 (1:200, eBioscience) antibodies for 30 min on ice. Next, the cells were washed twice with fluorescent-activated cell sorting buffer (2% fetal calf serum and 2 mM ethylenediaminetetraacetic acid in PBS), and analyzed using an LSRFortessa (BD Biosciences, San Jose, CA, United States) flow cytometer. The flow cytometric data were analyzed using FlowJo software version 10 (BD Biosciences).