Legs of age-matched male WT and Ostf1lacZ/LacZ sibling mice were isolated. Bone marrow from tibia and femurs were expelled from bone shafts using a MEM-containing syringe and 23 gauge needle. Subsequently, bone marrow was triturated to dissociate the cells and then plated in MEM/FCS/Pen-Strep/Glutamine containing macrophage colony stimulating factor (M-CSF, 100 ng/ml, Prospec) to induce macrophage production. After 2 days, M-CSF dependent macrophages were dissociated and 5000 cells were put in each well of a Lab-Tek multichamber. Medium was then changed to MEM/FCS/Pen-Strep/Glutamine containing 25 ng/ml of M-CSF and 100 ng/ml of Receptor activator of nuclear factor kappa-B ligand (RANKL, Prospec) to induce osteoclast differentiation. Cells were left to differentiate for 5 days before processing.
M csf
Manufactured by ProSpec
Sourced in Israel, United States
M-CSF is a recombinant protein that functions as a hematopoietic growth factor. It stimulates the survival, proliferation, and differentiation of monocytes, macrophages, and their precursor cells.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by ProSpec (5 mentions)
Penicillin, by Merck Group (4 mentions)
Raw 264.7 macrophage like cells atcc, by LGC (4 mentions)
Rankl, by ProSpec (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Il 13, by ProSpec (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Lithium heparin coated blood tubes, by BD (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Il 13, by Thermo Fisher Scientific (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
M csf, by Thermo Fisher Scientific (2 mentions)
Macs ls column, by Miltenyi Biotec (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Rankl, by R&D Systems (2 mentions)
Tgf β1, by ProSpec (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
28 protocols using m csf
1
Osteoclast Differentiation from Bone Marrow
2
Isolation and Culture of Murine Macrophages
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BMDMs were isolated from 6 to 8-week-old BABL/c male mice as described (Davis, 2013 (link)). Primary cells were maintained on bacterial grade plates for 1 week in DMEM (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, 100 units/mL penicillin G, 100 mg/mL streptomycin (Invitrogen) and 5 ng/mL M-CSF (ProSpec). Adherent cells were then replated on plastic tissue culture plates in fresh media and used 24 h after replating. The immortalized rat Kupffer cell line (purchased from Applied Biological Materials Inc.) was maintained in the same media as BMDMs but without M-CSF.
For stimulation, BMDMs or the Kupffer cell line were exposed to recombinant IFN-γ, IL-10, IL-4 or IL-13 (ProSpec) after starvation overnight in 0.5% fetal bovine serum (Hyclone), and harvested at the appropriate time points.
For transfection, 40 nM miRNA mimics, inhibitors or negative control (GenePharma, China) was transfected using the Lipofectamine 2000 Transfection Reagent (Life technologies) according to the manufacturer's instructions.
For stimulation, BMDMs or the Kupffer cell line were exposed to recombinant IFN-γ, IL-10, IL-4 or IL-13 (ProSpec) after starvation overnight in 0.5% fetal bovine serum (Hyclone), and harvested at the appropriate time points.
For transfection, 40 nM miRNA mimics, inhibitors or negative control (GenePharma, China) was transfected using the Lipofectamine 2000 Transfection Reagent (Life technologies) according to the manufacturer's instructions.
3
Bone Marrow-Derived Macrophage Isolation
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BALB/c mice at the age of 6–8 weeks were purchased from Animal Research Laboratories, Himberg, Austria. The femora and tibiae of the mice were removed after scarifying and bone marrow cells were collected. Bone marrow cells were seeded at 1 × 105 cells/cm2 into 12-well plates and grown for 5–7 days in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% of 10,000 units penicillin and 10 mg streptomycin/mL (Sigma, St Louis, MO, USA) and with 20ng/mL mouse macrophage colony-stimulating factor (M-CSF; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel). RAW 264.7 macrophage-like cells (ATCC; LGC Standards GmbH, Wesel, Germany), expanded in regular DMEM growth medium without supplement. Macrophages were also prepared from Nrf2 knockout and wildtype mice that were kindly provided by T. Kensler, Johns Hopkins University, Baltimore, MD, USA, and M. Yamamoto, University of Tsukuba, Tennoudai, Japan. Human gingival fibroblasts were prepared by explant cultures after approval of the Ethical Committee of the Medical University of Vienna (EK Nr. 631/2007). All cells were cultured under standard conditions at 37 °C, 5% CO2, and 95% humidity.
4
Isolating Peritoneal Macrophages from Mice
5
Isolation of Bone Marrow-Derived Macrophages
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BMDMs were isolated from C/EBPδ deficient and wild-type mice, as described before [17 (link)]. In detail, tibia and femur of naïve wild-type and C/EBPδ−/− mice were harvested, and bone marrow was flushed out of the bones with RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L -glutamine, 100 units/mL penicillin and 500 μg/mL streptomycin (all Lonza, Basel, Switzerland). Erythrocytes were lysed by incubation in erythrocyte lysis buffer for 10 minutes at room temperature after which the remaining cells were plated in a non-tissue culture treated petri dish in complete RPMI medium containing 30 ng/mL M-CSF (Macrophage-colony stimulating factor; CYT-43; ProSpec, Rehovot, Israel) in order to allow macrophage differentiation. After 7 days, cells were harvested using 4 mg/mL lidocaine in 5 mM EDTA/PBS and immediately used for stimulation experiments.
6
Generation of DCs and Macrophages from BM
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DCs and macrophages were prepared according to a modified protocol described by Lutz et al. (1999) (link). In brief, 2 × 106 BM cells from tibia and femur of SPLF and SPLMx1KO mice were cultured in RPMI 1640, 10% heat-inactivated FBS, β-ME, glutamine, and penicillin/streptomycin supplemented with 100–200 ng/ml GM-CSF (Prospec) for DCs or 100–200 ng/ml M-CSF (Prospec) for macrophages for 6 or 7 d. DCs were matured for one additional day with 100 ng/ml LPS (Sigma-Aldrich).
7
Differentiation of Human Monocytes
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Human CD14+ primary Mo were isolated from freshly prepared buffy coat (Karolinska Institutet Biobank, Stockholm, Sweden) using a pluriBead cell separation kit (pluriSelect GmbH, Leipzig, Germany). Mo were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) in six-well culture plates at a density of 1 × 106/ml and exposed to NhhA, as indicated, to induce differentiation. Control cells were primed with M-CSF (50 ng/ml) for 5 days. A subset of these cells was then polarized for 24 h by either IFN-γ (10 ng/ml) or LPS (20 ng/ml; Sigma-Aldrich, St. Louis, MO) to M1Mφ or IL-4 (20 ng/ml) to M2Mφ. To generate DCs, Mo were cultured in GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) for 5 days followed by LPS (20 ng/ml) for 2 days to enhance maturation. M-CSF and GM-CSF were purchased from ProSpec-Tany TechnoGene Ltd. (Israel). IL-4, IFN-γ, and IL-13 were purchased from ImmunoTools (Germany).
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
8
Murine Bone Marrow-Derived Macrophages Isolation
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For the isolation and culture of murine bone marrow-derived macrophages, 6–8 week old BALB/c mice (Animal Research Laboratories, Himberg, Austria) were purchased. Bone marrow cells were collected from the femora and tibiae and grown for 5 days in Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and antibiotics (Sigma Aldrich, St. Louis, MO, USA) supplemented with 20 µg/mL macrophage colony-stimulating factor (M-CSF; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel). RAW 264.7 macrophage-like cells (LGC Standards, Wesel, Germany) were expanded in growth medium and seeded at 1 × 106 cells/cm2 into 24-well plates (CytoOne, Starlab International, Hamburg, Germany). The cells were cultured in the growth medium at 37 °C, 5% CO2, and 95% humidity. Serum-free conditions were used during cell stimulation.
9
Dendritic Cell Cytokine Culture
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Recombinant human cytokines used for DC treatments were as follows: LT-α (TNF-β), GM-CSF, M-CSF, IL-1β, IL-6, IL-4, TNF-α, IL-13, IFN-γ, IL-10 (ProSpec-Tany Technogene, Rehovot, Israel) and TGF-β1 (R&D Systems, Minneapolis, Minnesota, USA) were used in culture within a final concentration range of 5–80 ng/ml. Dexamethasone was used at a final concentration in culture at 30 ng/ml (Sigma-Aldrich, St. Louis, MO). All cell culture experiments utilized RPMI 1640 tissue culture medium, heat-inactivated (56°C/20 min) fetal calf serum (FCS), penicillin/streptomycin and L-glutamine (SAFC Biosciences, Lenexa, KS).
10
Isolation and Differentiation of Murine Bone Marrow-Derived Macrophages and Osteoclasts
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Bone marrow cells were flushed out of the long bones of the mice and the cell suspension cultured in αMEM supplemented with 10% FCS, Pen/Strep and 100 ng/ml M-CSF (Prospec Bio) for three days. Next the non-adherent cells were removed by washing the cultures with PBS, and the adherent BMDMs harvested using Acutase (Sigma) or cell dissociation buffer (Enzyme-Free; PBS-based) (Gibco, Life Technologies). For osteoclast formation experiments, cells were plated in 96-well plates at 15 × 103 cells in 100 µl medium supplemented with 25 ng/ml M-CSF and up to 100 ng/ml RANKL (a kind gift of Dr. J. Dunford, University of Oxford) per well. The culture medium was refreshed at day 2 and day 4 and the cells fixed on day 5 with 4% buffered formalin and stained for TRAP. TRAP positive cells containing three or more nuclei were counted as osteoclasts. For RNA isolation, BMDM cells were seeded in 6-well plates at 5 × 105 cells per well in medium supplemented with 25 ng/ml M-CSF only (for macrophage cultures) or 25 ng/ml M-CSF plus 100 ng/ml RANKL (for osteoclast cultures) and cultured for 5 days as described above. For Western Blot analysis, BMDM cells were seeded in 6-well plates at 5 × 105 cells per well in medium supplemented with 25 ng/ml M-CSF and cultured for 3 days.
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