The tibias and femurs from 8-week-old male C57BL/6 mice were extracted, and the bone
marrow was flushed as described previously43 (link). Bone marrow mononuclear cells (BMNCs) were then isolated by density-gradient
centrifugation with Ficoll-Paque PREMIUM 1.084 (GE Healthcare, Little Chalfont, United
Kingdom). CD133+ cells were separated from BMNCs with anti-mouse-prominin-1
microbeads (Miltenyi MACS; Miltenyi Biotec, Auburn, CA, USA) using a magnetically
activated cell sorter (Miltenyi Biotec). Freshly isolated cells were seeded at
2×105 cells/well in 24-well plates (NEST, China) pre-coated with 0.015 mg/ml
poly-L-lysine (PLL; Sigma-Aldrich) and vitronectin (Gibco, Invitrogen, Carlsbad, CA, USA).
The cells were then cultured in alpha minimal essential medium (αMEM) supplemented with
10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin (Gibco), 100
ng/ml stem cell factor, 100 ng/ml FMS-like tyrosine kinase-3 ligand, 20 ng/ml
interleukin-6 and 20 ng/ml leukemia inhibitory factor (PeproTech, Rocky Hill, NJ, USA)
according to previous studies with slight modifications39,44–46 at 37°C in a
humidified atmosphere containing 5% CO2. The cells were harvested with 0.05%
trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) before passaging.
marrow was flushed as described previously43 (link). Bone marrow mononuclear cells (BMNCs) were then isolated by density-gradient
centrifugation with Ficoll-Paque PREMIUM 1.084 (GE Healthcare, Little Chalfont, United
Kingdom). CD133+ cells were separated from BMNCs with anti-mouse-prominin-1
microbeads (Miltenyi MACS; Miltenyi Biotec, Auburn, CA, USA) using a magnetically
activated cell sorter (Miltenyi Biotec). Freshly isolated cells were seeded at
2×105 cells/well in 24-well plates (NEST, China) pre-coated with 0.015 mg/ml
poly-L-lysine (PLL; Sigma-Aldrich) and vitronectin (Gibco, Invitrogen, Carlsbad, CA, USA).
The cells were then cultured in alpha minimal essential medium (αMEM) supplemented with
10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin (Gibco), 100
ng/ml stem cell factor, 100 ng/ml FMS-like tyrosine kinase-3 ligand, 20 ng/ml
interleukin-6 and 20 ng/ml leukemia inhibitory factor (PeproTech, Rocky Hill, NJ, USA)
according to previous studies with slight modifications39,44–46 at 37°C in a
humidified atmosphere containing 5% CO2. The cells were harvested with 0.05%
trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) before passaging.
