BMMs were cultured with density of 10000 cells/well in a 96-well plate. RANKL (50 ng/ml) without or with Vericiguat (0, 100 μM, 500 nM, 1 μM, 2 μM, 4 μM, and 8 μM) was added at the second day for 6 d. Recombinant mouse-derived M-CSF and RANKL were purchased from Novoprotein Scientific Inc. (Pudong New District, China). The identification of OC was evaluated by TRAP staining using a tartrate resistant acid phosphatase staining kit (Sigma-Aldrich Institute of Biotechnology, St. Louis, MO, USA). TRAP-positive cells were observed and imaged using light microscopy (Nikon, Tokyo, Japan).
M csf
Manufactured by Novoprotein
Sourced in China
M-CSF is a recombinant protein that functions as a macrophage colony-stimulating factor. It is a hematopoietic growth factor that promotes the survival, proliferation, and differentiation of monocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Rankl, by Novoprotein (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cck 8 kit, by Dojindo Laboratories (2 mentions)
Raw264, by American Type Culture Collection (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Penicillin streptomycin, by Novoprotein (2 mentions)
Ros assay kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Vazyme (1 mentions)
Liperfluo, by Dojindo Laboratories (1 mentions)
Ferrostatin 1, by MedChemExpress (1 mentions)
Thioglycolate, by BD (1 mentions)
α mem, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Solarbio (1 mentions)
Interleukin 4 (il 4), by Sino Biological (1 mentions)
Rosiglitazone, by MedChemExpress (1 mentions)
Gw9662, by MedChemExpress (1 mentions)
Rankl, by Corning (1 mentions)
M csf, by Corning (1 mentions)
Penicillin and streptomycin, by Solarbio (1 mentions)
22 protocols using m csf
1
Osteoclast Differentiation with Vericiguat
2
Ferroptosis Molecular Assays Protocol
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Prussian-blue kit (Solarbio, Beijing, China), ROS Assay kit (Beyotime, Shanghai, China), Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China), CCK-8 kit, and Liperfluo (Dojindo, Shanghai, China) were used following instructions. All ELISA kits were purchased from Dakewe (BioLegend, California, USA). Ferrostatin-1 (HY-100579), Liproxtatin-1 (HY-12726) and RSL3 (HY-100218A) was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA) and prepared in DMSO. M-CSF (Novoprotein, Shanghai, China) for BMDM was prepared in ddH2O. NIR-797 and Rhodamine B (Hualanchem, Shanghai, China) were prepared in DMSO. The antibodies used for flow cytometry (supplemental Table S1 ) and PCR Assay plates (Wcgene Biotech, Shanghai, China) were all purchased from fcmacs (Nanjing, China). SPIO nanoparticles (Ferumoxytol) were kindly provided by professor Ning Gu from Southeast University.
3
Differentiation of Bone Marrow-Derived Macrophages
4
Isolation of Mouse Macrophages
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To obtain bone marrow-derived macrophages, bone marrow cells were isolated from mouse tibia and femur, and then cultured in 10 cm dish with DMEM containing 50 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF; Novoprotein, China). After 2 days of culture, the supernatant was replaced with DMEM containing 50 ng/mL M-CSF. The cells were allowed to grow for additional 4–5 days. The adherent macrophages were collected for further experiments.
For isolation of mouse peritoneal macrophages, 4% Thioglycolate (BD bioscience, USA) was intraperitoneally injected into 6-week-old C57BL/6 mice. 4 days later, mice were sacrificed, peritoneal macrophages were collected by peritoneal cavity lavage with 8–10 mL DMEM. After centrifugation at 5000 rpm for 5 min, macrophages were resuspended for further experiments.
For isolation of mouse peritoneal macrophages, 4% Thioglycolate (BD bioscience, USA) was intraperitoneally injected into 6-week-old C57BL/6 mice. 4 days later, mice were sacrificed, peritoneal macrophages were collected by peritoneal cavity lavage with 8–10 mL DMEM. After centrifugation at 5000 rpm for 5 min, macrophages were resuspended for further experiments.
5
Murine Macrophage Polarization and Osteoclastogenesis
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Murine macrophage lines RAW264.7 were purchased from American Type Culture Collection (ATCC, USA) and maintained in α-MEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Lonsera, Uruguay) and 1% antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml ) providing atmosphere of 37℃ and 5% CO2. In order to research macrophages polarization, cells were treated with different concentrations of AGEs (Bioss, China), 1μg/ml LPS (Solarbio, China) and 20ng/ml IL-4 (Sino Biological, China). To induce macrophages to differentiate into osteoclasts, α-MEM containing 50ng/ml RANKL (Novoprotein, China) and 30ng/ml M-CSF (Novoprotein, China) was used to culture cells for 6 days. The rst 3 days of osteoclasts differentiation were considered to be the early stage, and the last 3 days were considered to be the late stage. Three repeat holes were set for each sample.
6
Macrophage Activation by SARS-CoV-2 and R848
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Primary human macrophages were obtained by plating fresh or frozen splenocytes obtained from donors with ruptured spleen caused by trauma with RPMI 1640 medium with 50 ng/mL M-CSF (Novoprotein, Suzhou, Jiangsu, China) and removing unattached cells after 12 hours of culturing. The attached macrophages were then cultured in complete RPMI-1640 medium or RPMI-1640 medium without glutamine and pyruvate (Seahorse Bioscience, Santa Clara, California, USA) for 12 h before simulation with R848 (Sigma, St. Louis, Missouri, USA) or SARS-CoV-2 (MOI = 1). In other experiments, macrophages were treated with 2-DG (1mM; Sigma, St. Louis, Missouri, USA), PPARγ inhibitor GW9662 (2μM; MedChemExpress, Monmouth Junction, NJ, USA), or PPARγ activator rosiglitazone (5μM; MedChemExpress, Monmouth Junction, NJ, USA) for 48 h before stimulation with R848 or SARS-CoV-2 as indicated (MOI = 1). After 24 h of stimulation, supernatants were collected for the measurement of IL-6, TNF-α, CCL2, CCL3 by ELISA and cells were harvested for the detection of IL1B, CD36, and FABP4 mRNA by real-time PCR.
7
Osteoclastogenesis and Bone Resorption Assay
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For the osteoclastogenesis assay, BMCs were isolated from the mouse monocyte depletion model as mentioned above. Cells were cultured for 5–6 days in α-modified Eagle’s medium (α-MEM, HyClone, Logan, Utah, USA) containing 10% FBS, 1% penicillin–streptomycin (Invitrogen), receptor activator of nuclear factor kappa B ligand (RANKL) (25 ng/ml, Novoprotein, Suzhou, China), and macrophage colony-stimulating factor (MCSF) (40 ng/ml, Novoprotein) in an incubator under 5% CO2 at 37 °C35 (link),38 (link). Cells were fixed and stained for TRAP. Digital images were acquired using a light microscope.
For the bone resorption assay, cells were plated on Corning Osteo Assay Surface 24-well plates (Corning, Corning, NY, USA) and cultured in 40 ng/ml MCSF and 25 ng/ml RANKL for 14 days. Cells were removed using an ultrasonic cleaner, and resorption pits were captured using a light microscope (Leica).
For the bone resorption assay, cells were plated on Corning Osteo Assay Surface 24-well plates (Corning, Corning, NY, USA) and cultured in 40 ng/ml MCSF and 25 ng/ml RANKL for 14 days. Cells were removed using an ultrasonic cleaner, and resorption pits were captured using a light microscope (Leica).
8
Isolation and Cultivation of Murine Bone Marrow-Derived Macrophages
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Mice were sacrificed and soaked with 75% alcohol for 10 minutes for disinfection. The bone marrow cavities of the leg bones were opened, flushed with DMEM (GNM12800, Genom). The supernatants were passed through 200-mesh filter and the cell suspension was collected after centrifugation for 5 minutes at 500 Â g 4 C. Then cells were incubated in ACK lysis buffer (Cat. #R1010, Solarbio) for 5 minutes to remove the red blood cells. Cells were cultured in DMEM (GNM12800, Genom) with 10% (vol/vol) FBS (Cat. #10270, Gibco) supplemented with 2% (vol/vol) penicillin and streptomycin (Cat. #P1400, Solarbio) and 20 ng/mL mouse macrophage colony stimulating factor (M-CSF; Cat. #CB34, Novoprotein). DMEM containing M-CSF was renewed every 2 days until macrophages were induced. Six days later, bone marrow-derived macrophages (BMDM) were confirmed by flow cytometry for F4/80 (Cat. #565411, Clone T45-2342, 1 mg/mL, BD Biosciences).
9
Cell Culture Protocols for HEK293T, RAW264.7, and BMDMs
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HEK293T cells and RAW264.7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2 in a humidified incubator.
BMDMs were cultured as described (Liu et al. 2011 (link)). Briefly, bone marrow cells from the femurs of mice were seeded into plates in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (Novoprotein). After three days, fresh medium was added to the plate. The cells were ready for use on day 6.
BMDMs were cultured as described (Liu et al. 2011 (link)). Briefly, bone marrow cells from the femurs of mice were seeded into plates in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (Novoprotein). After three days, fresh medium was added to the plate. The cells were ready for use on day 6.
10
Osteoclastogenesis Regulation Pathway
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M-CSF (#CB34) and RANKL (#CR06) were both acquired from Novoprotein (Shanghai, China). FBS (#E01021) was purchased from Evacell (Hong Kong, China). Alpha-modified MEM (α-MEM) and penicillin‒streptomycin were obtained from HyClone (USA). Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).
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