The bone marrow of the femur and tibia of 4-week-old wild-type male mice were washed to collect monocytes and macrophages (BMMs). The washed bone marrow cells were cultured in α-MEM culture media containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 30 ng/mL M-CSF (# 416-ML-010/CF, R&D, United States) for 6 h. After the adherent cells were discarded, the floating cells were incubated with α-MEM culture media containing M-CSF (30 ng/mL) to obtain pure BMMs. When the BMMs were incubated in a 12-well plate (2 × 105 cells/well) containing 30 ng/mL M-CSF and 50 ng/mL RANKL (# 462-TEC-010, R&D), all cells became precursor osteoclasts after 3 days of culture. The incubation was continued with 30 ng/mL M-CSF and 100 ng/mL RANKL for 7 days to form mature multinucleated osteoclasts. Then, the conditioned media was harvested from preosteoclasts and mature osteoclasts. After centrifugation (2,500 r.p.m., 10 min at 4°C), the conditioned medium was aliquoted and stored at −80°C. In subsequent experiments, neutralizing antibodies for certain factors, including IGFBP5 (# sc-515116, Santa Cruz, 1 μg/mL), CXCL12 (# sc-74271, Santa Cruz, 1 μg/mL), and CTGF (# sc-365970, Santa Cruz, 1 μg/mL), were added to the conditioned media for 3d (CFE) or for 7d (chondrogenesis).
M csf
Manufactured by R&D Systems
Sourced in United States, United Kingdom, China, Germany, France, Japan, Israel, Switzerland, Canada
M-CSF is a recombinant human protein that functions as a cytokine. It is involved in the differentiation and proliferation of monocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Rankl, by R&D Systems (225 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (104 mentions)
Gm csf, by R&D Systems (56 mentions)
α mem, by Thermo Fisher Scientific (53 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (43 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (43 mentions)
Interleukin 4 (il 4), by R&D Systems (39 mentions)
Streptomycin, by Thermo Fisher Scientific (36 mentions)
Penicillin, by Thermo Fisher Scientific (35 mentions)
Ifn γ, by R&D Systems (32 mentions)
Rankl, by Thermo Fisher Scientific (32 mentions)
Lipopolysaccharides (lps), by Merck Group (32 mentions)
M csf, by Thermo Fisher Scientific (28 mentions)
Fetal bovine serum (fbs), by Merck Group (25 mentions)
Rpmi 1640, by Thermo Fisher Scientific (25 mentions)
Trap staining kit, by Merck Group (22 mentions)
α mem, by R&D Systems (15 mentions)
L glutamine, by Thermo Fisher Scientific (15 mentions)
Leukocyte acid phosphatase kit, by Merck Group (14 mentions)
L glutamine, by Merck Group (13 mentions)
827 protocols using m csf
1
Osteoclast Differentiation and Conditioned Media
2
Osteoclastogenesis from Bone Marrow Macrophages
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BMMs were obtained as described previously [66 (link)]. Briefly, whole bone marrow cells were cultured with 10% FBS, 1% PSG, 10 ng/mL of M-CSF (R&D Systems, Minneapolis, MN, USA) for 12 h. Non-adherent bone marrow cells were then replated in Petri dishes with 10 ng/mL M-CSF and adherent BMMs were harvested as osteoclast precursors 4–5 days later. To generate pre-osteoclasts or mature osteoclasts, BMMs were cultured with 30 ng/mL M-CSF and 30 ng/mL RANKL (R&D Systems) for 3 or 5 days at 37 °C. To enumerate osteoclasts, the cells were stained with tartrate-resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphatase Assay Kit following the manufacturer’s instructions (Sigma-Aldrich). A giant osteoclast formation was identified as a multinucleated (>10 nuclei) TRAP-positive cell.
3
Bone Marrow-Derived Macrophage Differentiation
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BMDM were differentiated as described previously17 (link). Bone marrow cells from WT mice were cultured in the presence of M-CSF (20 ng ml−1, R&D Systems) in a petri dish (BD OPTILUX). After 7 days, the macrophages (1 × 107 per 10 ml) were stimulated with IL-4 (30 ng ml−1, R&D Systems), IL-13 (30 ng ml−1, R&D Systems), IL-33 (30 ng ml−1, BioLegend) (M2 differentiation); or LPS (1 μg ml−1, E. coli 0111:B4, Sigma-Aldrich), IFN-γ (200 ng ml−1, R&D Systems), M-CSF (5 ng ml−1, R&D Systems) (M1 differentiation); or M-CSF (5 ng ml−1, R&D Systems) alone (M0 macrophages) for 2 days. In some experiments, macrophages were cultured with 10 ng ml−1 IL-4 (R&D Systems) and/or 20 ng ml−1 IL-33 (BioLegend) for 2 days. At the end of cultures, the cells were used for FACS and supernatants for cytokine analysis by ELISA.
4
Osteoclast Differentiation from Bone Marrow Cells
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Total bone marrow cells were harvested as described above. After red blood cells were removed using ACK buffer (0.01 mM EDTA, 0.011 M KHCO3, and 0.155 M NH4Cl, pH 7.3), the remaining cells were cultured with 10% FBS, 1% PSG, and 10 ng/ml M-CSF (R&D Systems) for 24 h. Non-adherent cells were cultured in Petri dishes, with 30 ng/ml M-CSF, for 4–5 days to generate BMMs. BMMs were cultured with 30 ng/ml of M-CSF and 30 ng/ml of RANKL (R&D Systems) for 4–5 days to generate osteoclasts. To enumerate osteoclasts, cells were fixed with 10% neutral buffered formalin for 10 min and stained for tartrate-resistant acid phosphatase (TRAP), using the Leukocyte Acid Phosphatase Assay Kit, following the manufacturer’s instructions (Sigma-Aldrich). An osteoclast was defined as a multinuclear (more than 3 nuclei) TRAP-positive cell.
5
Osteoclast Differentiation from Bone Marrow Macrophages
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Osteoclasts were differentiated from bone marrow macrophages as described [13 (link)]. Briefly, BMCs collected from the femurs and tibias of 8 week-old ddY mice were cultured in α-MEM containing 10% FBS and macrophage colony-stimulating factor (M-CSF, 10 ng/mL; R & D Systems, Minneapolis, MN) for 3 days. The bone marrow macrophages were cultured in M-CSF alone or M-CSF plus RANKL (10 ng/mL; R & D Systems) with or without rhinacanthin C for 3 days. In experiments testing the effects of rhinacanthin C on the LPS-induced osteoclastogenesis, BMMs were primed with RANKL(1 ng/mL) for 24 h before LPS treatment.
6
Osteoclast Differentiation Protocol
7
Differentiation of Macrophages from Monocytes
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CD14+ monocytes were purified from peripheral blood mononuclear cells (PBMCs) of healthy donors using CD14 MicroBeads (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions as previously described [25 (link)]. Approval for these studies was granted by the National Institute of Allergy and Infectious Diseases Institutional (NIAID) Review Board. Participants provided informed written consent before blood was drawn. To generate M-CSF-induced macrophages (mMac) or AB macrophages (Ab), the isolated CD14+ monocytes were cultured for seven days in the presence of 25 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA) in macrophage serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and 5 μg/mL of Gentamycin) or 10% human AB serum (Geminin bioproducts, West Sacramento, CA, USA), respectively. IL-27-induced macrophages (iMac or Abi) were generated from the CD14+ monocytes by culturing for seven days in the presence of 25 ng/mL of M-CSF or 10% AB serum with 100 ng/mL of IL-27 (R&D Systems). The culture media was changed with fresh media every 3–4 days. All induced macrophages (Ab/Abi and mMac/iMac) cells were maintained in D-MEM medium (Thermo Fisher Scientific) containing 10% FBS (HyClone Laboratories, Logan, UT), 10 mM HEPES, and 5 μg/mL Gentamicin.
8
Osteoclast Differentiation and TRAP Staining
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Osteoclast differentiation and TRAP staining
were carried out as previously reported.25 (link) Bone marrow cells (BMCs) were isolated in strict accordance with
the Standard Protocol for Animal Study of Sunchon National University
(SCNU). The animal use protocol was approved by the Institutional
Animal Care and Use Committee of SCNU (Permit No. SCNU IACUC 2021-05).
All efforts were made to minimize suffering. In short, bone marrow
cells (BMCs) were isolated by flushing femurs and tibias of 5-week-old
ICR mice.25 (link) The isolated BMCs were cultured
in α-MEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen
Life Technologies, Grand Island, NY) with 10 ng/mL M-CSF (R&D
Systems) for 1 day. Bone marrow macrophages, obtained by removing
floating cells, were used for osteoclast differentiation. BMMs (1
× 104 cells/well) were cultured with M-CSF (30 ng/mL)
and RANKL (10 ng/mL; R&D Systems) for 3 days to generate preosteoclasts.
The preosteoclasts were treated with vehicle or indicated compounds
in the presence of M-CSF (30 ng/mL) for 30 min and then cultured with
RANKL (10 ng/mL) to differentiate into mature tartrate-resistant acid
phosphatase-positive multinucleated cells (TRAP+-MNCs).
After 1 day, the cells were fixed with 3.7% formaldehyde for 5 min,
permeabilized with 0.1% Triton X-100 for 5 min, and stained using
the leukocyte acid phosphatase kit 387-A.
were carried out as previously reported.25 (link) Bone marrow cells (BMCs) were isolated in strict accordance with
the Standard Protocol for Animal Study of Sunchon National University
(SCNU). The animal use protocol was approved by the Institutional
Animal Care and Use Committee of SCNU (Permit No. SCNU IACUC 2021-05).
All efforts were made to minimize suffering. In short, bone marrow
cells (BMCs) were isolated by flushing femurs and tibias of 5-week-old
ICR mice.25 (link) The isolated BMCs were cultured
in α-MEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen
Life Technologies, Grand Island, NY) with 10 ng/mL M-CSF (R&D
Systems) for 1 day. Bone marrow macrophages, obtained by removing
floating cells, were used for osteoclast differentiation. BMMs (1
× 104 cells/well) were cultured with M-CSF (30 ng/mL)
and RANKL (10 ng/mL; R&D Systems) for 3 days to generate preosteoclasts.
The preosteoclasts were treated with vehicle or indicated compounds
in the presence of M-CSF (30 ng/mL) for 30 min and then cultured with
RANKL (10 ng/mL) to differentiate into mature tartrate-resistant acid
phosphatase-positive multinucleated cells (TRAP+-MNCs).
After 1 day, the cells were fixed with 3.7% formaldehyde for 5 min,
permeabilized with 0.1% Triton X-100 for 5 min, and stained using
the leukocyte acid phosphatase kit 387-A.
9
Osteoblast and Osteoclast Differentiation
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MSCs were isolated from the bone marrow of healthy donors and cultured in osteoblast differentiation medium to obtain mature osteoblasts [20 (link)]. The bone formation activity of osteoblasts was demonstrated using Alizarin red S staining (Sigma-Aldrich, Cat# A5533). Human monocytes were isolated from peripheral blood mononuclear cells of health donors and cultured in M-CSF (25 ng/ml) (R&D Systems, Cat# 216-MC) for 7 days to obtain the precursors of osteoclasts. The precursors derived from human monocytes were cultured in M-CSF (25 ng/ml) and RANKL (10 ng/ml) (R&D Systems, Cat# 6449-TEC), cocultured with or without E. coli or LPS for 7 days to induce mature osteoclast formation. Leukocyte acid phosphatase kit (Sigma-Aldrich, Cat# 387A) for TRAP staining was used to detect mature osteoclasts.
10
Mouse Macrophage and Kidney Cell Culture
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The mouse macrophage cell line RAW264.7 (ATCC, Cat# TIB-71™) and human kidney epithelial cell line 293T (ATCC, Cat#CRL-3216™) were cultured in DMEM supplemented with 10% foetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. Primary mouse BMM was isolated from 3- to 4-week-old male Balb/c mice as previously described [22 (link)]. Briefly, femurs were dissected under aseptic conditions, the bone marrow cavity was flushed with complete α-MEM containing 10% FBS and 1% penicillin-streptomycin, and a single-cell suspension was prepared. Cells were cultured in complete α-MEM at 37°C in a 5% CO2 atmosphere overnight. M-CSF (R&D Systems, Cat# 416-ML-010) was added to a final concentration of 50 ng/ml, and the cells were cultured for an additional 3 days. For OC differentiation, cells were cultured in complete α-MEM containing 50 ng/ml M-CSF and 50 ng/ml RANKL (R&D Systems, Cat# 462-TEC-010) for 6 days. For drug treatment, 10 nM SBI-0206965 (MCE, Cat# HY-16966) or 20 nM LYN-1604 dihydrochloride (MCE, Cat# HY-101923B) was used to inhibit or activate ULK1, respectively.
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