M2 macrophages were generated from monocytes of healthy donors, as previously described [61 (link)]. Informed consent was received from all donors. Monocytes were isolated from leukocyte retaining systems obtained from blood donations at the Institute for Transfusion Medicine (Kiel, Germany). First, peripheral blood mononuclear cells (PBMC) were isolated from leukocyte enriched blood donations via density gradient centrifugation. Subsequenly, monocytes were isolated from PBMC via counterflow centrifugation. Fractions with a monocyte purity of >90% were used for macrophage differentiation. For this purpose, 15 × 106 monocytes were resuspended in RPMI-1640 medium supplemented with 1% FCS, 2 mM L-glutamine, 1% penicillin-streptomycin (all purchased from PAA, Pasching, Austria), and 50 ng/mL recombinant human macrophage colony-stimulating factor (M-CSF) (Bio-Legend, Fell, Germany), seeded into VueLife cell culture bags (CellGenix, Freiburg, Germany) and differentiated for 7 days at 37°C, 5% CO2, and 86% humidity into macrophages according to established protocols. The phenotype of anti-inflammatory M2-macrophages was validated as described previously.
Recombinant human macrophage colony stimulating factor m csf
Manufactured by BioLegend
Sourced in Austria, United States
Recombinant human macrophage colony-stimulating factor (M-CSF) is a cytokine that stimulates the production, differentiation, and function of macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by BioLegend (1 mentions)
Vuelife cell culture bags, by CellGenix (1 mentions)
Tunicamycin, by Cayman Chemical (1 mentions)
Rankl, by BioLegend (1 mentions)
Tib 202, by American Type Culture Collection (1 mentions)
Monocyte attachment medium, by PromoCell (1 mentions)
Easysep human cd14 positive selection kit, by STEMCELL (1 mentions)
Anti human cd3, by BioLegend (1 mentions)
Anti human cd28, by BioLegend (1 mentions)
Easysep t cell enrichment kit, by STEMCELL (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
C3h hen mice, by Japan SLC (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Raw264.7 murine macrophage like cell line, by InvivoGen (1 mentions)
L glutamine, by Corning (1 mentions)
6 protocols using recombinant human macrophage colony stimulating factor m csf
1
Generation of M2 Macrophages from Monocytes
2
Macrophage Differentiation and Activation
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Recombinant human macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL) and mouse RANKL were purchased from BioLegend (San Diego, CA, United States). Tunicamycin was obtained from Cayman Chemical Co. (Ann Arbor, MI, United States).
3
Differentiation and Polarization of Macrophages
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Unless otherwise specified, folate‐free RPMI 1640 medium containing 10% of heat‐inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen) was used for all cell culture studies. THP‐1 cells (ATCC, TIB‐202™) were differentiated into macrophages as described by Genin et al (2015 ) and then polarized to M2‐like macrophages as outlined by Fernando et al (2014 ).
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood and cultured for 2 h in monocyte attachment medium (PromoCell) at a density of 1 million/cm2, after which the cells were washed 4× with pre‐warmed PBS and differentiated for 7 days into unpolarized macrophages in RPMI medium containing 20 ng/ml of recombinant human macrophage colony‐stimulating factor (M‐CSF) (BioLegend). Polarization of the resulting macrophages into M2‐like macrophages was conducted for 48 h as described above.
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood and cultured for 2 h in monocyte attachment medium (PromoCell) at a density of 1 million/cm2, after which the cells were washed 4× with pre‐warmed PBS and differentiated for 7 days into unpolarized macrophages in RPMI medium containing 20 ng/ml of recombinant human macrophage colony‐stimulating factor (M‐CSF) (BioLegend). Polarization of the resulting macrophages into M2‐like macrophages was conducted for 48 h as described above.
4
Isolation and Differentiation of Macrophages and T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density centrifugation from fresh whole blood donated by healthy volunteers. Human monocytes were purified from PBMCs by EasySep Human CD14 Positive Selection Kit (StemCell Technologies; Cat No.17858) and human naïve CD3+ T cells were purified by EasySep T cell enrichment kits (StemCell Technologies; Cat No. 19751). The purity of monocytes and naïve T cells were > 97%, as confirmed by flow cytometry. CD14+ monocytes were maintained in RPMI-1640 supplemented with 10% FBS and 100 ng/mL recombinant human macrophage colony stimulating factor (M-CSF) (Biolegend; Cat No. 574802) for 7 days to generate macrophages. For induction of TAMs, macrophages were seeded in 12-well plates and stimulated with 1ml mixture of CM and FBS-containing medium (1:1) for 24 h. Human naïve T cells were first activated using anti-human CD3 (Biolegend; Cat No.317326) pre-coated 6-well plates and then cultured with RPMI-1640 containing 10% FBS, 1 μg/mL anti-human CD28 (Biolegend; Cat No.302914) and 100 IU/mL recombinant human IL-2 (Pepro Tech; Cat No.2000250).
5
Murine Bone Marrow Macrophage Differentiation
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Femoral and tibial bone marrows of 12‐ to 16‐week‐old nondiabetic C3H/HeN mice (Japan SLC Inc.) were collected in 1% FCS‐supplemented low‐glucose DMEM, and erythrocytes were lysed in RBC lysis buffer. Bone marrow cells were washed in 1% FCS DMEM and cultured 5–7 days in 10% FCS DMEM supplemented with 40 ng/ml recombinant human macrophage colony‐stimulating factor (M‐CSF) (BioLegend). Non‐adhesive cells were washed out with PBS. Differentiated macrophages were collected by scraping in ice‐cold PBS with 1% FCS/ 5 mM EDTA, washed, counted, diluted to 2.0 × 105 cells/ml in 1% FCS DMEM, and cultured in 96‐well plates overnight at 37°C under 5% CO2 atmosphere.
6
Primary Human Macrophage Differentiation
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Isolated peripheral blood primary human monocytes were purchased from STEMCELL Technologies. For in vitro differentiation of monocytes into human macrophages, isolated monocytes were cultured in complete RMPI 1640 supplemented with 10% heat-inactivated FBS (PAA, GE Healthcare), 2 mM l -glutamine (Corning), and 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific) in the presence of recombinant human macrophage colony-stimulating factor (M-CSF) (50 ng/ml) (BioLegend) for 7 days. The RAW264.7 murine macrophage-like cell line (InvivoGen) and the THP-1 monocytic cells derived from an acute monocytic leukemia patient cell line (American Type Culture Collection) were cultured at 37°C in a 5% CO2 humidified atmosphere. All cells were grown and maintained in DMEM (Gibco; Thermo Fisher Scientific) with 10% heat-inactivated FBS (PAA, GE Healthcare),and 100 U of penicillin-streptomycin (Gibco; Thermo Fisher Scientific). The culture medium was replaced every 3 days, and upon reaching 80% confluency, cultures were passaged. RAW264.7 cell passaging was achieved by gentle cell scraping and seeding cells at a density of 3 × 106 cells per T75 flask (Nunc; Thermo Fisher Scientific).
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