Dendritic cells were generated from bone marrow progenitors, as described elsewhere (Lewis et al., 2008b (link)). Briefly, bone marrow was prepared from femurs and tibias of donor mice. Cells were seeded at 3 × 103 cells per culture plate, in 10 ml RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM l -glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. Medium was added 10 ng/ml recombinant granulocyte macrophage colony stimulating factor (GM-CSF, Prospec, Rehovot, Israel). After 8 days, BMDC entered in vitro assays.
Gm csf
Manufactured by ProSpec
Sourced in Israel, United States
GM-CSF is a recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). It is a cytokine that functions to stimulate the production and differentiation of granulocytes and macrophages from bone marrow precursor cells.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by ProSpec (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
M csf, by ProSpec (5 mentions)
Streptomycin, by ProSpec (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
M csf, by Thermo Fisher Scientific (3 mentions)
Ifn γ, by ProSpec (3 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
X vivo 15 media, by Lonza (3 mentions)
Facscanto 2, by BD (3 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Il 1β, by ProSpec (2 mentions)
Il 13, by ProSpec (2 mentions)
Interleukin 6 (il 6), by ProSpec (2 mentions)
Il 10, by ProSpec (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)
31 protocols using gm csf
1
Dendritic Cell Generation from Murine Bone Marrow
2
Generating Murine Bone Marrow-Derived Dendritic Cells
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Mouse bone marrow-derived dendritic cells (DCs) were generated, as described previously (23 (link)). In brief, the femurs and tibiae of 8-week-old female BALB/c mice were cut and flushed out with ice-cold RPMI 1640 using a syringe. Thereafter, cells were dissociated by pipetting and then filtered using a 70 μm cell strainer to remove debris. The RBCs were lysed using RBC lysis buffer. Lastly bone marrow cells (5 × 106 cells/10 ml) were seeded in petri dishes with complete RPMI 1640 containing 10 ng/ml GM-CSF (ProSpec, Rehovort, Israel) and cultured at 37°C in a 5% CO2 incubator. The fresh RPMI1640 with GM-CSF was added on day 3 and day 5. At day 7, loosely adherent cells were harvested and seeded in 24-well plate at 2 × 106 cells/well. Then, the cells were stimulated with heat-inactivated 14BME20 or lipopolysaccharide (LPS) (100 ng/ml; Sigma-Aldrich) for 24 h.
3
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).
4
Differentiating MUTZ-3 Cells into Dendritic Cells
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The human leukemic cell line MUTZ-3 was obtained from the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and cultured at 37 °C with 5% CO2 in Minimal Essential Medium-α (MEM-α) (Gibco, Life Technologies, Paisley, UK) supplemented with 20% heat-inactivated fetal calf serum (FCS) (Lonza, Basel, Switzerland), 1% penicillin/streptomycin and 500 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (ProsPec; Rehovot, Israel). Cells from passage numbers 10 to 25 were used for differentiation to a DC phenotype. Differentiation conditions tested are summarized in Appendix (Table A1 ). The culture medium was replaced every 3 days. Cells were seeded at a density of 2 × 105 cells/mL in 12-well plates (Corning, New York, NY, USA) for flow cytometry analysis.
5
Monocyte-derived Dendritic Cell Differentiation
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Monocytes were purified from PBMCs that were isolated from healthy donors using Ficoll-Plaque (GE Healthcare UK, Buckinghamshire, UK) by positive selection with anti-human CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and transduced with lentiviral vectors in the presence of polybrene (8 ng/ml; Sigma, St. Louis, MO). The cells were cultured in α-MEM (Wako, Tokyo, Japan) containing 20% FCS (Nichirei Bioscience, Tokyo, Japan), 50 ng/ml of M-CSF (ORF Genetics, Kopavogur, Iceland), and 50 ng/ml of GM-CSF (Prospec, East Brunswick, NJ). After 4–5 weeks, proliferating cells (CD14-ML) appeared [16 (link)]. To induce differentiation of CD14-ML into DC, the cells were cultured in the presence of M-CSF (50 ng/ml), GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) for 3 days. To induce the maturation, CD14-ML-DC were stimulated with penicillin-killed Streptococcus pyogenes (10 μg/ml, OK432; Chugai Pharmaceutical, Tokyo, Japan) for 2 days.
6
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.
7
Differentiation and Stimulation of BMDCs and BMDMs
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B16-F1 (Riken BRC, Ibaraki, Japan) cells were grown in D-MEM (High Glucose, 043-30085, FUJIFILM Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS). RAW 264.7 (Riken BRC) cells were grown in MEM (Minimum Essential Medium Eagle, M4655, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS. BMDCs were generated as described previously (Kanemaru et al., 2017 (link)). Briefly, mouse bone marrow cells were obtained from femurs and differentiated into DCs in MEM with 10% FBS containing 10 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; Prospec, East Brunswick, NJ, USA). Medium was replaced with fresh medium containing GM-CSF every two days. On day 6, cells were collected. BMDMs were also generated as described previously (Kanemaru et al., 2017 (link)). Bone marrow cells were obtained and incubated in MEM with 10% FBS containing 3 ng/ml macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA). Medium was changed on day 3. On day 5, macrophages were harvested. BMDCs and BMDMs were stimulated with LPS (100 ng/ml; LPS-SM ultrapure, InvivoGen, San Diego, CA, USA) and/or L-(+)-Lactic acid (Sigma-Aldrich), and/or sodium L-lactate (Sigma-Aldrich).
8
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).
9
Monocyte-Derived Macrophage Differentiation
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Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Paque (GE Healthcare, Chicago, IL, USA) density centrifugation from whole blood collected from a single healthy volunteer (following institutional standard operating protocol and under the approval of Medical Ethics Review Committee) in lithium-heparin coated blood tubes (BD, Franklin Lakes, NJ). For monocyte isolation from PBMC, the MACS Monocyte Isolation kit with CD14+ magnetic beads was used with MACS LS Columns according to the manufacturer’s manual (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated monocytes were plated in fully supplemented RPMI media and treated with either 50 ng/ml M-CSF (Prospec, Rehovot, Israel) or 50 ng/ml GM-CSF (Prospec) for 6 days to obtain M0 subtype monocyte-derived macrophages (MDM); media were refreshed together with the cytokines every 2 days. To obtain the M1 and M2 MDM subtypes, all media were removed on day six, after which the M0 macrophages were treated for 24 h with 50 ng/ml IFN-γ (Peprotech) on the previously GM-CSF treated group or with 20 ng/ml IL-4 (Peprotech) and IL-13 (Peprotech) on the previously M-CSF treated group to obtain M1 and M2 MDMs respectively. Conditioned media from all subtypes were collected and processed as for the THP-1 derived macrophage media.
10
Differentiation of Macrophage Subtypes
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Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R&D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). A mouse monoclonal antibody (clone 3C9) that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, San Francisco, CA). RPMI 1640 tissue culture medium, foetal calf serum (FCS) and L-glutamine were purchased from SAFC Biosciences (Lenexa, KS).
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