Primary bone marrow derived macrophages were obtained [46 (link)] from bone marrow aspirate collected from femurs and tibiae of 7 week old C57BL/6 mice (Charles River Laboratories) or B6.129P2(SJL)-MyD88/J mice (Jackson Laboratories) and placed in medium containing Iscove’s Modified Dulbecco’s Medium (IMDM), 20% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 2.5 μg/ml fungizone (PSF) and layered in Lympholyte M (Accurate Chemicals). Mononuclear cells were plated on non-TCPS and differentiated into macrophages in IMDM, 20% FBS, 2 mL-glutamine, in PSF, 1.5 ng/ml human macrophage colony stimulating factor (R&D systems) and 100 ng/ml human FLT-3 (R&D systems) for 10 days. Hydrogels and the reference substrate, TCPS, were exposed to FBS for two hours at room temperature to allow proteins to adsorb; the FBS was then removed by aspiration. Macrophages were seeded on the surface of equilibrium swollen PEG hydrogels or on the reference substrate with pre-adsorbed serum proteins at 2,650 macrophages/mm2 under: (a) no treatment, (b) 10 ng/ml lipopolysaccharide (LPS, Sigma), or (c) 1 μg/ml poly(I:C) (InvivoGen) in medium containing IMDM, 20% FBS, and PSF.
Human macrophage colony stimulating factor
Manufactured by R&D Systems
Sourced in United States, Germany
Human macrophage colony-stimulating factor (hM-CSF) is a recombinant cytokine that promotes the survival, proliferation, and differentiation of macrophages. It is a glycoprotein that binds to the CSF-1 receptor and stimulates the production and function of macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Poly 1 c, by InvivoGen (1 mentions)
B6.129p2 sjl myd88 j mice, by Jackson ImmunoResearch (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Eagle s minimum essential medium, by Merck Group (1 mentions)
Penicillin, by R&D Systems (1 mentions)
Streptomycin, by R&D Systems (1 mentions)
Ficoll hypaque lymphoprep, by Abbott (1 mentions)
1640 medium, by Lonza (1 mentions)
Human serum, by Merck Group (1 mentions)
Human m csf, by R&D Systems (1 mentions)
Rpmi 1640, by R&D Systems (1 mentions)
Cd14 monocytes, by AllCells (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Cd14 monocytes, by Lonza (1 mentions)
Rpmi 1640, by Nacalai Tesque (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
M csf, by R&D Systems (1 mentions)
Trap kit, by Merck Group (1 mentions)
Easysep human monocyte isolation kit, by STEMCELL (1 mentions)
6 protocols using human macrophage colony stimulating factor
1
Macrophage Differentiation and Activation on PEG Hydrogels
2
Osteoclast Differentiation from Spleen Cells
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Spleen cells were isolated from 4- to 6-week-old WT or src−/− mice and cultured under 5% CO2 at 37 °C in alpha modification of Eagle's minimum essential medium from Sigma-Aldrich supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) with human macrophage colony-stimulating factor (30 ng/ml) from R&D Systems Inc for 3 days, and then, the cells (100,000 cells/well, in a 24-well plate) were replated on culture plates from Corning, coverslips (Fisher Scientific), or dentin slices and cultured with human soluble RANKL (100 ng/ml) from R&D Systems Inc for 5 days. Cells were infected with adenoviruses expressing cre recombinase (CRE) (multiplicity of infection [MOI] 50) and Src family kinases or chimeras (MOI 50) on day 4 after replating and culture for 1 day.
3
Isolation and Differentiation of Human Monocyte-derived Macrophages
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Human PBMCs from healthy volunteers were isolated from heparinized venous blood using Ficoll-Hypaque (Lymphoprep; Alere technologies, Oslo, Norway) as described previously.18 (link) For monocyte-derived macrophages (MDMs) differentiation, adherent monocytes were incubated in Roswell Park Memorial Institute 1640 medium (Lonza, Basel, Switzerland) containing 5% pooled human serum (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine, for 1 hour at 37°C, after which the nonadherent cells were removed. Human MDMs were prepared by culturing peripheral blood monocytes for 4 days in the presence of 4 ng/mL human macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). SARS-CoV-2 (2019-nCoV) NC-His recombinant protein (cat. No. 40588-V08B), Spike S1-His recombinant protein (cat. No. 40591-V08H), Spike S2 extracellular domain (ECD)-His recombinant protein (cat. No. 40590-V08B), and Spike RBD-His recombinant protein (cat. No. 40592-V08H) were purchased from Sino Biological, Beijing, China. Cells were stimulated with the proteins as indicated in figure legends.
4
Generating Human Monocyte-Derived Macrophages and Dendritic Cells
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Two types of human monocyte-derived macrophages (h-Mo-Mφs) were generated as described in Supplementary Figure S1A . CD14+ monocytes (AllCells, Alameda, CA, USA) were cultured (37°C, 5% CO2) in RPMI1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, Bergisch Gladbach, Germany) or 100 ng/ml human macrophage colony-stimulating factor (M-CSF; R&D Systems) for 7 days. The M-CSF-treated cells were further cultured in RPMI1640 supplemented with 10% FBS, 100 ng/ml human M-CSF, and 20 ng/ml human interleukin (IL)-4 (R&D Systems) for 2 days. Human monocyte-derived DCs (h-Mo-DCs) were obtained as shown in Supplementary Figure S2B . For h-Mo-DC generation, CD14+ monocytes were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 100 ng/ml human GM-CSF and 100 ng/ml human IL-4. Cells were incubated at 37°C in an atmosphere containing 5% CO2 for 6 days, with medium changes on days 2 and 3. Generated cells were harvested on day 6 and reseeded in fresh medium supplemented with 100 ng/ml GM-CSF and 100 ng/ml IL-4. To generate mature DCs, cells were further supplemented with 10 μg/ml CD40 agonist antibody (patent, WO/2002/088186). The characteristics of monocyte-derived cells were verified by flow cytometry (FCM).
5
Osteoclast Differentiation from Human Monocytes
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HOPs (CD14+ peripheral blood mononuclear cells) were isolated from normal human peripheral blood mononuclear cells using EasySep Human Monocyte Isolation Kit (StemCell Technologies) following the manufacturer’s instructions. HOPs were plated at 0.5×106 cells/well in a 24-well plate with 1 mL media (αMEM supplemented with 1 mm sodium pyruvate and 10% FBS). To stimulate osteoclast formation, 25 ng/mL of human macrophage colony-stimulating factor and 30 ng/mL of human RANKL (R&D Systems) were added. Cells were allowed to differentiate for 7 days with half media replaced every 3 days. On Day 5, Lec2 CHO cells (0.1×106 cells) sialylated as described above using glycosyl donor 10 were added to wells. Unlabeled Lec2 CHO cells were used as a negative control. On Day 7, cells were fixed and permeablized followed by staining for TRAP using the TRAP kit following manufacturer protocols (Sigma Aldrich). Osteoclasts were defined as TRAP+ with 3 or more nuclei.
6
Isolation and Polarization of Human Monocyte-Derived Macrophages
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Human PBMs from healthy volunteer donors were isolated by density‐gradient centrifugation using Ficoll–Hypaque (Pharmacia, Cat# IAE‐1). Harvested PBMs were seeded at a density of 2 × 106 cells/well per 24‐well plate in DMEM (Gibco, Cat#12430062) supplemented with 10% heat‐inactivated human AB serum (Gemini Bio‐Products, Cat#100–512), 50 U of penicillin per ml, 50 μg of streptomycin per ml, 2 mM l ‐glutamine, and 20 ng/ml human macrophage colony‐stimulating factor (R&D Systems, Cat#416‐ML) to stimulate macrophage differentiation. After 6 days of culture, nonadherent cells were removed by repeated gentle washing with a warm medium, and more than 95% of the adherent cells generated from current procedures were CD14+ monocytes/macrophages, indicating a good purity of monocytes/macrophages. For in vitro activation, monocyte‐derived macrophages at 2 × 106 cells were treated for 1 day with 45 ng/ml recombinant human interleukin‐4 (IL‐4; R&D Systems, Cat#204‐IL) for M2 polarization cell models or 25 μg/ml lipopolysaccharide (LPS; Sigma, Cat#LPS25) in order to generate M1 polarization cell models.
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