Ficoll paque premium 1

Macrophages were obtained by peritoneal lavage. Under sterile condition, SD rats (250–300 g) were intraperitoneally injected into 20‐mL RPMI medium and peritoneal exudate cells were then harvested. After centrifuged and suspended, total cell numbers were determined by Trypan blue staining. Cells were plated in 24‐well culture plates at 2×10⁵ cells/well in RPMI medium supplemented with 10% FBS. After 2 hours of incubation at 37°C and 5% CO₂, non‐adherent cells were washed away. The purity of macrophage was >90%, assessed by fluorescence‐activated cell sorting (FACS) analysis using anti‐CD68 (AbD Serotec, #MCA341) and anti‐granulocytes (BD Biosciences, #550002) (Figure S1B).
Neutrophils were obtained by density centrifugation over a Ficoll gradient (Ficoll‐Paque PREMIUM 1,084; GE Healthcare). After rats were anesthetized with 3% pentobarbital sodium (30 mg/kg) intraperitoneally, blood was taken by puncture of the left heart ventricle. Neutrophils were harvested according to the manufacturer's instructions. To assess the purity, positively selected neutrophils were made into cells smear. After being stained in Wright staining, 200 cells were performed by cell differential counts. Neutrophils were 91.3±1.2% (n=4). The purity of neutrophils was also >90%, assessed by FACS analysis using anti‐granulocytes and anti‐CD11b (Figure S1C).

The tibias and femurs from 8-week-old male C57BL/6 mice were extracted, and the bone
marrow was flushed as described previously^{43 (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 modifications^39,44–46 at 37°C in a
humidified atmosphere containing 5% CO₂. The cells were harvested with 0.05%
trypsin/ethylenediaminetetraacetic acid (EDTA; Invitrogen) before passaging.