Macrophages were derived from cultures of bone marrow cells (2×106 cells/mL) in non- treated plastic 100 mm plates with 10ng/mL recombinant M-CSF (Sigma-Aldrich, St. Louis, MO) in 10% FBS, DMEM (Gibco™) media with antibiotics (Gibco™). Media changes were performed at 3.5 days and at day 7. Macrophages were harvested with trypsin-EDTA treatment for 15 min at 37°C and seeded in 12 well plates for further polarization. Macrophages were stimulated for the next 24 h with or without LPS (10 ng/mL, Sigma) and murine IFN-γ (100 ng/mL, Sigma). Glucose was adjusted to 5 mM and 25 mM with the addition of 45% glucose solution (Gibco™). At the end of the experiment, cells were lysed and RNA was extracted as described in the methods.
Recombinant m csf
Manufactured by Merck Group
Sourced in United Kingdom, United States
Recombinant M-CSF is a laboratory product manufactured by Merck Group. It is a recombinant form of the human macrophage colony-stimulating factor (M-CSF) protein, which plays a role in the differentiation and proliferation of macrophages. The product is intended for research use only and not for use in diagnostic or therapeutic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glucose solution, by Thermo Fisher Scientific (1 mentions)
Murine ifn γ, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
5 azac, by Abcam (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258 dye, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg fitc antibody, by Merck Group (1 mentions)
Mts kit, by Promega (1 mentions)
Anti tubulin antibody, by Merck Group (1 mentions)
Anti vinculin antibody, by Merck Group (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
Parp 1, by Santa Cruz Biotechnology (1 mentions)
5 protocols using recombinant m csf
1
Macrophage Polarization Protocol
2
Isolation and Stimulation of Murine Bone Marrow-Derived Macrophages
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Bone marrow-derived macrophages (BMDM) were generated from murine bone marrow cells as described previously (19 (link)). Briefly, bone marrow cells were obtained from wild type (WT) and Nrf2 KO C57JBL/6 mice and cultured in DMEM medium containing growth factors of 20 ng/ml recombinant M-CSF (Sigma–Aldrich) and 10% FBS at 37°C, 5% CO2. The culture medium was replaced every 72 h. Adherent cells were collected after 6 days whereas non-adherent cells were removed by washing the plates with phosphate buffer solution (PBS) twice. Macrophages were enriched by positive selection with anti- F4/80 magnetic beads (Miltenyi Biotec). The enriched F4/80+ macrophages were typically of > 90% purity as determined by flow cytometry (Figure 1A
3
Bone Marrow-Derived Macrophage Isolation and Stimulation
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Bone marrow-derived macrophages were isolated from the marrow of femurs and tibias from 4-week-old male rats (100–150 g). Cells were then differentiated into BMDMs in Roswell Park Memorial Institute medium containing 10 ng/mL recombinant M-CSF (Sigma-Aldrich), 10% fetal bovine serum (Gibco, Paisley, UK), and streptomycin/penicillin (Gibco). Cell-culture population consisted of nearly 80% BMDMs at 7 days after seeding. The cells were harvested, washed with complete media, and plated in 6-well plates with or without 5-Aza-2′-deoxycytidine (5-aZac; 10 µmol/L; Abcam, Cambridge, UK) for 48 h. The cells without 5-aZac were stimulated with PBS, LPS (1 µg/mL), or LPS (1 µg/mL) + 1, 25(OH)2D3 (100 pg/mL) for 12 h, respectively, whereas cells with 5-aZac were stimulated with LPS (1 µg/mL) + 1, 25(OH)2D3 (100 pg/mL) for 12 h. After storage in liquid nitrogen, the cells and supernatant of five samples from four groups were transferred to a −80°C freezer, respectively, and then subsequently used for real-time PCR and ELISA analyses.
4
Murine Osteoclast Formation Assay
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Rob was purchased from ChemFaces (CAS No. 301‐19‐9, catalog number: CFN98375, purity≥98%, Wuhan ChemFaces Biochemical Co., Ltd., Wuhan, China) and dissolved in a concentration of 1 mM nuclease‐free water and DMSO. C57BL/6 mice for the cellular and animal experiments were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. The culture media, α‐MEM, HBSS, and FBS were obtained from Gibco (Carlsbad, CA, USA). Rm‐sRANKL was purchased from PeproTech (Rocky Hill, NJ, USA), and recombinant M‐CSF was obtained from Sigma‐Aldrich (St. Louis, MO, USA). Detailed information regarding the antibodies used is listed in the Table S2 .
5
Dracorhodin perchlorate modulates osteoclastogenesis
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Dracorhodin perchlorate (D.P) was obtained from Chengdu Must Bio‐Technology Co., Ltd, and was diluted in DMSO at a concentration of 100 mmol/L for storage, which was further diluted to the working dilutions with PBS. Alpha‐Minimum essential medium (α‐MEM, REF#12571‐063), foetal bovine serum (FBS, REF#16000‐044), DAPI (Cat#D3571), Hoechst 33258 dye (REF#H21491), rhodamine phalloidin (Cat#R415) and ProLong Gold Antifade Mountant (REF#P36970) were commercially available from Thermo Fisher Scientific. MTS kit was obtained from Promega (REF#G358A). Primary antibodies against Integrin β3 (Cat#sc‐6617‐R), MMP9 (Cat#sc‐21733), CTSK (Cat#sc‐48353), β‐actin (Cat#sc‐47778), IκB‐α (Cat#sc‐371), p65 (Cat#sc‐8008), PARP‐1 (Cat#sc‐8007) and NFATc1 (Cat#sc‐7294) were commercially available from Santa Cruz Biotechnology. Primary antibodies against c‐Fos (Cat#2250S), p‐p65 (Cat#3031S), p‐JNK (Cat#9251L) and JNK (Cat#9252L) were purchased from Cell Signalling Technology. Antimouse IgG‐FITC antibody (Cat#F9137), anti‐vinculin antibody (Cat#V9264), anti‐tubulin antibody (Cat#T3526) and recombinant M‐CSF (Cat#M6518) were commercially available from Sigma‐Aldrich. Recombinant GST‐rRANKL was produced and purified as previously described.26
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