BMSCs were isolated using a previously reported method [14 (link)]. In brief, the mouse femur was obtained, and the marrow cavity was washed with culture medium. The washing fluid was centrifuged and resuspended, and cells were inoculated into the culture dish. BMSCs were identified by flow cytometry, and antibodies used for flow cytometry were: PE anti-mouse/human CD44 (BioLegend, 103007, 1: 20), APC anti-mouse stem cell antigen 1 (Sca-1; BioLegend, 108111, 1: 80), PerCP/Cyanine5.5 anti-mouse CD34 (BioLegend, 128608, 1: 20), and PE/Cyanine7 anti-mouse CD45 (BioLegend, 103114, 1: 80). All experiments involved cells at passages 3–5.
Bone marrow-derived macrophages (BMDMs) were isolated and treated according to a previous method [15 ]. Typically, BMDMs were isolated using the same method of BMSCs isolation. The cell number was determined after resuspension, and the cell concentration was adjusted to 0.5×106/ml. Then, 20 ng/ml macrophage colony stimulating factor (M-CSF; PeproTech, 315-02-10, America) was added to the culture medium. The nonadherent cells were removed after 3 days, and fresh M-CSF-containing culture medium was added. Four days later, the mature macrophages were harvested for subsequent experiments.
Bone marrow-derived macrophages (BMDMs) were isolated and treated according to a previous method [15 ]. Typically, BMDMs were isolated using the same method of BMSCs isolation. The cell number was determined after resuspension, and the cell concentration was adjusted to 0.5×106/ml. Then, 20 ng/ml macrophage colony stimulating factor (M-CSF; PeproTech, 315-02-10, America) was added to the culture medium. The nonadherent cells were removed after 3 days, and fresh M-CSF-containing culture medium was added. Four days later, the mature macrophages were harvested for subsequent experiments.
