M csf 216 mc

HVSMCs (HASMCs, HCASMCs, and HPASMCs) were cultured in Smooth Muscle Cell Growth Medium-2 (CC-3182; Lonza, Basel, Switzerland). Human pluripotent stem cell-derived immortalized monocytic cell lines (MLs) were cultured as previously described [13 (link)] in StemPro-34 serum-free medium (10639011; Thermo Fisher Scientific) containing 2 mM of l-glutamine and 50 ng/mL of M-CSF (216-MC; R&D Systems).

We used 3-week-old C57BL/6J mice for cell extraction and obtained approval from the Animal Ethics Committee of Qilu Hospital, Shandong University (DWLL-2021-136). After administering Pelltobarbitalum Natricum (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) to euthanize the mice, we carefully isolated the femur and flushed the bone marrow cavity using the basal medium. We used RPMI-1640 and DMEM/F12 (1:1) medium to culture BMDMs and BMMSCs respectively. 10% Fetal Bovine Serum (FBS), and 1% antibiotics were added to the basal medium. Bone marrow cells were cultured for 7 days to obtain BMDMs by adding 50 ng/mL M-CSF (#216-MC, R&D System, Minneapolis, MN, USA) to RPMI-1640 as previously described [18 (link)]. Subsequently, 20 ng/ml IL4 was added and incubated for 24 hours to induce differentiation of BMDMs to M2 macrophages. We examined M2 macrophage differentiation using flow cytometry. Place the cells in the incubator and set the temperature to 37°C. The medium was changed regularly and passaged after digestion using trypsin (#25200056, Gibco, Rockville, MD, USA). Cells within 5 generations were selected for the experiment.

Human peripheral blood mononuclear cells were obtained from heparinized peripheral blood by gradient density centrifugation using Ficoll-Paque medium. Monocytes were purified by nonmonocyte depletion with antibody-conjugated magnetic-activated cell sorting microbeads (MACS II Monocyte Isolation Kit; Miltenyi Biotec). Cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (2917354; MP Biomedicals, Santa Ana, CA, USA), 50 mg/ml streptomycin, and 50 U/ml penicillin. To differentiate cells into M2- or M1-like Mϕ, 50 ng/ml macrophage colony-stimulating factor (M-CSF) (216-MC; R&D Systems) or 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (215-GM; R&D Systems) was added, respectively [25 (link)]. Cells were incubated for 10 consecutive days, and medium change was performed at days 2, 5, and 8. For some experiments, M1- and M2-like Mϕ were stimulated with lipopolysaccharide (LPS) (1 μg/ml, serotype 0111:B4; InvivoGen, San Diego, CA, USA), interferon (IFN)-α2b (1 U/μl; PBL Assay Science, Piscataway, NJ, USA), or IFN-β (1 U/μl; PBL Assay Science).