Biotin conjugated rat antibodies specific for mouse cd45

The tibiae and femurs were dissected from mice immediately after death. Total bone marrow cells were flushed from the bones, using a 23‐gauge needle and syringe, into ice‐cold FACS buffer containing CaCl₂‐ and MgCl₂‐free 1X PBS (Thermo Fisher Scientific, Carlsbad, CA, USA) and 2% FBS. Cells from individual mice in each group were centrifuged at 450 g for 6 min at 4 °C. After the red blood cells were removed with RBC lysis buffer (0.9% NH₄Cl with 20 mm Tris base, pH 7.4), bone marrow cells were suspended in ice‐cold FACS buffer. Cells were then incubated with biotin‐conjugated rat antibodies specific for mouse CD45 (eBioscience, San Diego, CA, USA; 14‐0451, 1:100). The labeled hematopoietic cells were depleted 3 times by incubation with anti‐rat IgG Dynabeads (Invitrogen, Grand Island, NY, USA) at a bead:cell ratio of approximately 4:1. Cells binding the Dynabeads were removed with a magnetic field. The negatively isolated CD45⁻ cells were washed twice and suspended with ice‐cold FACS buffer at 1–2 × 10⁶ cells mL⁻¹. Osx1‐TdRFP⁺ cells were sorted in an Aria II cell sorter (BD Bioscience, San Jose, CA, USA) using the PE‐A fluorochrome gate.

Osx1-TdRFP⁺ cells from mouse bone marrow were isolated as described before^{13 (link)}. Briefly, bone marrow cells were flushed from tibiae and femora into ice-cold FACS buffer and red blood cells removed with RBC lysis buffer (BD Bioscience). Cells were then incubated with biotin-conjugated rat antibodies specific for mouse CD45 (eBioscience, 14-0451, 1:100) and hematopoietic cells depleted with anti-rat IgG Dynabeads (Invitrogen). CD45^- Osx1-TdRFP⁺ cells were sorted in an Aria II cell sorter (BD Bioscience) using the PE-A fluorochrome gate.