Expansion of Mouse Bone Marrow Cells

The mouse BM cells (2 × 10⁷–3 × 10⁷) were flushed from the long bones with 3% fetal bovine serum (FBS) in PBS. A single-cell suspension of all nuclear cells was obtained by passing all BM cells through a 70-μm cell strainer (Bioscience, Dümmer, Germany). Then, 2.5 × 10⁵ cells per square centimeter were seeded into 10-cm culture dishes (Corning, Lowell, MA, USA) and incubated at 37 ℃ in 5% CO₂. After 48 h, the cultures were washed with PBS to eliminate the non-adherent cells. The attached cells were cultured for 10–15 d with α-modified essential medium (α-MEM; Gibco-BRL, Gaithersburg, MA, USA) supplemented with 20% FBS (Gibco-BRL), 2 mmol⋅L⁻¹ L-glutamine, 100 U⋅mL⁻¹ penicillin, and 100 mg⋅mL⁻¹ streptomycin (Invitrogen, Gaithersburg, MD, USA). The cell culture protocol and surface marker identification have been described in our previous studies^{16 (link),58 (link)} (Supplementary Table 1).