PBMCs were isolated from heparinized peripheral blood from healthy donors by density gradient centrifugation on Lymphoprep (Nycomed). For DC, macrophage, or LC culture, monocytes were isolated using Percoll (Pharmacia) density gradients. Cells were cultured for 6 days in IMDM (Lonza) containing 5% fetal bovine serum (FBS; Biowest) and 86 μg/mL gentamicin (Gibco) supplemented with 20 ng/mL recombinant human GM‐CSF (Invitrogen) and 2 ng/mL recombinant human IL‐4 (Miltenyi Biotec) for DCs, 20 ng/mL recombinant human GM‐CSF for macrophages, or 20 ng/mL recombinant human GM‐CSF, 2 ng/mL recombinant human IL‐4 and 10 μg/mL recombinant human TGF‐β1 (R&D Systems) for LCs. At day 2 or 3, half of the medium was replaced by fresh medium containing cytokines. Monocytes were isolated from PBMCs by MACS isolation using CD14 microbeads (Miltenyi Biotec) for direct stimulation or analysis.
Recombinant human gm csf
Manufactured by R&D Systems
Sourced in United States
Recombinant human GM-CSF is a cytokine that stimulates the production and function of granulocytes and macrophages. It is produced in a human cell expression system.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by R&D Systems (13 mentions)
Recombinant human il 4, by R&D Systems (11 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Collagenase d, by Roche (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Ifn γ, by R&D Systems (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Murine il 3, by Thermo Fisher Scientific (2 mentions)
Recombinant human ifn γ, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Detection kit, by Lonza (2 mentions)
Fetal bovine serum (fbs), by R&D Systems (2 mentions)
Dmem base medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 base medium, by Thermo Fisher Scientific (2 mentions)
40 m cell strainer, by BD (2 mentions)
Cd1a microbeads, by Miltenyi Biotec (2 mentions)
54 protocols using recombinant human gm csf
1
Isolation and Culture of Myeloid Cells
2
Recombinant Proteins for Immune Cell Study
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HCVc (amino acids2–192) and β-galactosidase was obtained from meridian life science Inc (Saco, USA). Human recombinant GM-CSF, M-CSF, IFN-γ, Lipopolysaccharide (LPS), IL-4, IL-13, Pam3CSK4 and anti-TLR2 antibody were purchased from R&D (NewYork, USA).
3
Monocyte-derived Dendritic and Langerhans Cell Generation
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Peripheral human blood of normal donors was obtained by buffy coats from the German Red Cross blood donation service, Berlin. All studies performed were in adherence to the Helsinki guidelines. By depletion of contaminating cells monocytes were magnetically isolated from PBMC (monocyte isolation kit II, Miltenyi Biotec, Bergisch Gladbach, Germany). In some studies, MoDCs and MoLCs were generated from the same donor blood. Cells were cultured in RPMI 1640 supplemented with 2 mM l -glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin and 10% (v/v) heat-inactivated FCS (Biochrom, Berlin, Germany) for 6 days with human recombinant GM-CSF (100 ng/mL), IL-4 (10 ng/mL), and TGF-β1 (for MoLCs only, 10 ng/mL, R&D Systems, Wiesbaden-Nordenstadt, Germany) [49 (link)].
4
Monocyte-Derived Dendritic Cell Generation
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Human monocytes were purified from peripheral blood lymphocytes were isolated by using Ficoll gradients (lympholyte-H; Cedarlane, Burlington, Ontario). CD14 cells were purified by positive sorting through anti-CD14 mAb-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in 75 cm2 flasks (Costar, Corning Life Sciences, Tewksbury, MA) in RPMI 1640 medium (Life Technologies Invitrogen), supplemented with heat-inactivated 10% lipopolysaccharide-free FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (all from EuroClone, Milan, Italy) and 0.05 mM 2-βmercaptoethanol (Sigma Chemicals; St. Louis, MO) in the presence of human recombinant GM-CSF (25 ng/ml; R&D Systems, Minneapolis, MN) and IL-4 (25 ng/ml; R&D Systems) [21] (link). After 6 d, immature MDDC were washed and analyzed by cytofluorimetry for the expression of the surface markers CD1a, CD14, CD83 and CD38. MDDC were used in the experiments if >80% CD1a and <10% CD14 positive cells.
5
Netrin-1 and RvD1 Modulate Macrophage Phagocytosis
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For differentiation of Mϕ, peripheral blood monocytes were isolated from human leukapheresis collars from the Children’s Hospital Boston Blood Bank and cultured in RPMI with 10 ng/ml human recombinant GM-CSF (R&D Systems; 37°C for 7 d). To prepare apoptotic PMN, human PMNs obtained from peripheral blood were isolated and labeled with carboxyfluorescein diacetate (10 µM, 30 min at 37°C; Invitrogen) and allowed to undergo apoptosis in serum-free RPMI for 16–18 h. MΦ (0.106 cells/well) were incubated with human recombinant netrin-1 (R&D Systems) in the presence or absence of RvD1 (1 nM) Boc2 peptide (10−4 M; Santa Cruz Biotechnology, Inc.; 15 min at 37°C). Apoptotic PMNs were added at 1:3 ratio (MΦ/PMN) with phagocytosis incubations performed at 37°C for 60 min, and fluorescence was determined by using a fluorescent plate reader (SpectraMax; Molecular Devices).
6
Monocyte-Derived Dendritic Cell Generation
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Fifty to 70 ml of blood was taken by venous puncture. Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-Paque (GE Healthcare) purification. Briefly, blood was diluted 1:1 with sodium chloride and tubes were centrifuged at 400g for 30 minutes with the brake off. Cells were harvested from the interface of the Ficoll layer, and were washed and enumerated. Monocytes were isolated using anti-CD14 microbeads, according to the manufacturer’s instruction (Miltenyi Biotec, San Diego, CA, USA). Cells were cultured in RPMI 1640 with 10% human serum and L-glutamine, pen/strep, non-essential amino acids, sodium pyruvate, 2-ME, 40 ng/ml of recombinant human IL-4 and 40 ng/ml of recombinant human GM-CSF (both from R&D Systems). Cytokines were replenished on days 3 and 6, and cells were used on day 7. For some experiments, CD4+ T cells were isolated from the CD14- fraction using the T cell isolation II kit (Miltenyi Biotec). Cells were then cultured with MoDCs in the presence or absence or RSV. At 48 hours, RNA was extracted and message levels of IFN-γ, IL-5 and IL-13 were determined by qPCR.
7
Engineered IDH2 Mutant Expression in TF-1 Cells
8
Cell Culture Conditions for K562, HEL92.1.7, and TF-I
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K562, HEL92.1.7, and TF-I cell lines were purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 2 mM l -glutamine (Sigma-Aldrich) 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO2 in the air. Additionally, TF-I cells medium contained 2 ng/ml of recombinant human GM-CSF (R&D Systems). Cells have been cultured for no longer than 4 weeks after thawing and were regularly tested for Mycoplasma contamination using PCR technique and were confirmed to be negative.
9
Differentiation of Monocytes into Macrophages and Induced Microglia
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Peripheral blood was collected using a heparinized tube from healthy adult volunteers and a patient of NHD. Peripheral blood mononuclear cells (PBMC) were isolated by Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. PBMC were resuspended with RPMI-1640 (Nacalai Tesque, Kyoto, Japan), 10% heat-inactivated fetal bovine serum (FBS; Endotoxin = 0.692 EU/ml; Japan Bio Serum, Hiroshima, Japan) and 1% antibiotics/antimycotic (Invitrogen, Carlsbad, CA). PBMC were plated onto culture chambers at a density of 4 × 105 cells/ml and cultured overnight in standard culture conditions (37°C, 5% CO2). After overnight incubation, culture supernatant and non-adherent cells were removed. The adherent cells (monocytes) were cultured with RPMI-1640 Glutamax (Invitrogen) supplemented with 1% antibiotics/antimycotic and a mixture of the following candidate cytokines; recombinant human GM-CSF (10 ng/ml; R&D Systems, Minneapolis, MN), recombinant human IL-34 (100 ng/ml; R&D Systems) and M-CSF (10 ng/ml; Peprotec, Rocky Hill, NJ) in order to develop iMG cells. We also developed induced macrophage from human monocytes; monocytes were cultured with RPMI-1640 Glutamax supplemented with 1% antibiotics/antimycotic and recombinant human GM-CSF (10 ng/ml). All cells were cultured in standard culture conditions for up to 14 days.
10
Culturing Monocyte-Derived Macrophages
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