Sitagliptin was purchased from Meilun Biotech (Dalian, China) and dissolved in normal saline. Soluble mouse recombinant M-CSF and RANKL were obtained from R&D Systems (Minneapolis, MN, United States). Fetal bovine serum (FBS), alpha-MEM and penicillin were purchased from Gibco BRL (Gaithersburg, MD, United States). Triton X-100, 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and TRAP staining solution were obtained from Sigma–Aldrich (St. Louis, MO, United States). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Kumamoto, Japan). The Acti-stain 555 fluorescent phalloidin was obtained from Cytoskeleton Inc. (Denver, CO, United States). The Cell Tracker Green, Fluo-4 AM, Hank’s balanced salt solution (HBSS), membrane dye DiI, and Pluronic F-127 were obtained from Life Technologies (Carlsbad, CA, United States). Primary antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States). Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay was purchased from Beyotime (Shanghai, China).
Soluble mouse recombinant m csf
Manufactured by R&D Systems
Sourced in United States
Soluble mouse recombinant M-CSF is a protein produced in a mouse cell expression system. It is a growth factor that stimulates the production and function of macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rankl, by R&D Systems (4 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Trap staining kit, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Nfatc1, by Cell Signaling Technology (3 mentions)
Alpha mem, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Mouse rankl, by R&D Systems (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Celltracker green, by Thermo Fisher Scientific (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Membrane dye dii, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Acti stain 555 fluorescent phalloidin, by Cytoskeleton (1 mentions)
7 protocols using soluble mouse recombinant m csf
1
Osteoclast Differentiation Assay
2
Osteoclast Differentiation Assay
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KF was obtained from Meilun (Dalian, Liaoning, China) and then it was dissolved in Dimethylsulfoxide (DMSO) with a concentration of 100 mM stock solution. Alpha-MEM, fetal bovine serum (FBS), and penicillin were purchased from Gibco BRL (Gaithersburg, MD, USA). Soluble mouse recombinant M-CSF and RANKL were purchased from R&D Systems (USA). Tartrate-resistant acid phosphatase (TRAP) staining solution was from Sigma–Aldrich. The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Japan). Primary antibodies targeting GADPH, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, NF-kB and IkB-a were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). The Prime Script RT reagent Kit and SYBR® Premix Ex Taq™ II were obtained from TaKaRa Biotechnology (Otsu, Shiga, Japan).
3
Osteoclastogenesis Assay with RAW264.7 and Primary BMMs
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TUS (purity >98%, Figure 3A
RAW264.7 cells were purchased from American Type Culture Collection (Rockville, MD, USA) and incubated in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin, namely complete α-MEM. C57BL/6 mice (4- to 6-week-old) were acquired from Shanghai Laboratory Animal Company (SLACCAS, Shanghai, China). Primary BMMs were separated from the whole bone marrow of murine femurs and tibias and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF. All cells used in this study were incubated at constant high humidity, 37°C, and 5% CO2 atmosphere (Zhang et al., 2018 (link)). Nonadherent cells were removed before each passage.
RAW264.7 cells were purchased from American Type Culture Collection (Rockville, MD, USA) and incubated in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin, namely complete α-MEM. C57BL/6 mice (4- to 6-week-old) were acquired from Shanghai Laboratory Animal Company (SLACCAS, Shanghai, China). Primary BMMs were separated from the whole bone marrow of murine femurs and tibias and cultured in complete α-MEM supplemented with 30 ng/ml M-CSF. All cells used in this study were incubated at constant high humidity, 37°C, and 5% CO2 atmosphere (Zhang et al., 2018 (link)). Nonadherent cells were removed before each passage.
4
Angelicin Modulates Macrophage Polarization
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Angelicin with purity > 95% was purchased from Med Chem Express (Monmouth Junction, NJ, United States) and dissolved in DMSO. Alpha Minimum Eagle’s Medium (alpha-MEM), penicillin (10,000 units/ml), streptomycin (10,000 mg/ml) and fetal bovine serum (FBS) were purchased from Hyclone (Waltham, MA, United States). Soluble mouse recombinant M-CSF was purchased obtained from R&D system (Minneapolis, MN, United States). Interleukin (IL)-4, IL-13, LPS, and IFN-γ were purchased from PeproTech Inc. (Rocky Hill, NJ, United States). The Cell Counting Kit-8 (CCK-8) was purchased from Med Chem Express (Monmouth Junction, NJ, United States). Anti-CD9, anti-gp130, anti-STAT3, anti-phospho-STAT3 (Ser727), anti-CD206, anti-iNOS, anti-Arg-1, anti-β-actin antibodies were purchased from Bioworld Technology (St. Louis Park, MN, United States).
5
Oxymatrine Regulates Osteoclastogenesis
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Oxymatrine was obtained from Selleck (Houston, Texas, United States). DMSO were obtained from Sigma-Aldrich (St. Louis, MO, United States). Recombinant soluble mouse M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, United States). DMEM (Dulbecco’s modified eagle medium), α-MEM (Eagle’s minimal essential medium with Alpha Modification), fetal bovine serum (FBS) as well as penicillin/streptomycin were all from Gibco-BRL. To prevent photosensitivity, the assays were all performed in the absence of visible light. To make DMSO content < 0.1%, OMT was diluted in cell culture medium, meanwhile, same concentration of DMSO used as a control group. Antibodies for ERK (extracellular signal-regulated kinase) (#4695), p38 (#8690), JNK (c-Jun N-terminal kinase) (#9252), phosphorylated p-ERK (Thr202/Tyr204) (#4370), p-p38 (Thr180/Tyr182) (#4511), p-JNK (Thr183/Tyr185) (#4668), NFATc1 (#8032) and GAPDH (#51332) were purchased from Cell Signaling Technology. TRAP (ab191406), Cathepsin K (ab187647), SREBP2 (ab30682) and NOX1 (ab131088) were purchased from Abcam. TRAP staining kit were obtained from Sigma-Aldrich.
6
Investigating IMD 0354 Effects on Osteoclast Formation
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IMD 0354 (Fig. 4a ) was purchased from Selleck Chemicals (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Eagle’s minimal essential medium with alpha modification (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco-BRL (Carlsbad, CA, USA). Cell Counting Kit-8 (CKK-8) was obtained from Dojindo Molecular Technology (Kumamoto, Japan). Recombinant soluble mouse M-CSF and mouse RANKL were obtained from R&D Systems (Minneapolis, MN, USA). IMD 0354 was dissolved in DMSO and stored at −20 °C. All experiments were performed in the absence of visible light to prevent photosensitivity. IMD 0354 was diluted in cell culture medium so that DMSO comprised <0.1% of the total volume. Specific antibodies against ERK, JNK, P38, phosphorylated p-ERK (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-P38 (Thr180/Tyr182), IKKα, IKKβ, p-IKKα/β, p-IκBα, IκBα, c-Fos, NFATc1, GAPDH, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). TRAP staining kit and all other reagents were purchased from Sigma-Aldrich, unless otherwise stated.
7
Pexmetinib Regulates Osteoclast Differentiation
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Pexmetinib was from Selleck Chemicals (Houston, Texas, USA). DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). α-MEM (Eagle’s minimal essential medium with Alpha Modification), dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco-BRL. CCK-8 (Cell Counting Kit-8) was obtained from Dojindo Molecular Technology (Kumamoto, Japan). Recombinant soluble mouse M−CSF, mouse RANKL and Colivelin were obtained from R&D Systems (Minneapolis, MN, USA). All experiments were performed in the absence of visible light to prevent photosensitivity. Pexmetinib was diluted in cell culture medium so that DMSO comprised < 0.1% of the total volume. Meanwhile, 0.1% DMSO as a control group in vitro. Specific antibodies against ERK (extracellular signal-regulated kinase, 1:1000), JNK (c-Jun N-terminal kinase, 1:1000), p38 (1:1000), phosphorylated p-ERK (Thr202/Tyr204, 1:1000), p-JNK (Thr183/Tyr185, 1:1000), p-p38 (Thr180/Tyr182, 1:1000), STAT3 (1:1000), p-STAT3 (Tyr705, 1:1000), NFATc1 (1:1000) and GAPDH (1:10000) were obtained from Cell Signaling Technology (Boston, USA), and antibodies against TRAP (1:1000), MMP-2 (1:1000) and MMP-9 (1:1000) were purchased from Abcam (Cambridge, UK). TRAP staining kit and all other reagents were purchased from Sigma-Aldrich, unless otherwise stated.
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