Mouse bone marrow-derived macrophages were isolated from femurs and matured for six days with macrophage colony-stimulating factor (M-CSF) containing L929 cell supernatant supplementation as described [46 (link)]. Human macrophages were matured under presence of 20 ng/ml recombinant human M-CSF (Miltenyi Biotec) for eight days from PBMC-derived monocytes sorted for CD14 expression by positive magnetic bead selection (Miltenyi Biotec) with purity >95%. Fresh buffy coats for PBMC isolation by gradient centrifugation (PAA) were obtained from the German Red Cross. For co-culture experiments, ABCB5+-derived MSCs or donor-matched ABCB5− HDFs were plated to adhere at 2×104 cells/well in 24-well plates in 0.5 ml DMEM with 10% high quality fetal bovine serum, 100 U/ml penicillin / streptomycin and 2 mM L-glutamine. After 24 h macrophages were seeded on top at 1×105 cells/-well in 0.5 ml, resulting in a 1:5 cell ratio unless indicated differently. Co-cultures were incubated with 50 U ml−1 recombinant mouse or human IFN-γ (R&D Systems) for 24 h and then stimulated with 20 ng ml−1 LPS (Sigma-Aldrich) and 50 U ml−1 IFN-γ for another 24 h period before supernatants were harvested and analyzed by ELISA (R&D Systems).
Recombinant human m csf
Manufactured by Miltenyi Biotec
Recombinant human M-CSF is a cytokine that stimulates the production, differentiation, and function of macrophages. It is a protein produced in a laboratory setting to mimic the naturally occurring human macrophage colony-stimulating factor.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic bead positive selection, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Forskolin, by Merck Group (1 mentions)
Pha from phaseolus vulgaris, by Merck Group (1 mentions)
As 605240, by Cayman Chemical (1 mentions)
Recombinant human il 4, by Miltenyi Biotec (1 mentions)
Recombinant human gm csf, by Miltenyi Biotec (1 mentions)
Pf 04418948, by Cayman Chemical (1 mentions)
Recombinant human tgf β1, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Imatinib mesylate, by Merck Group (1 mentions)
Mifepristone, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti lamp1 clone d2d11, by Cell Signaling Technology (1 mentions)
Macrophage serum free media, by Thermo Fisher Scientific (1 mentions)
Recombinant human ifn γ, by BD (1 mentions)
Lysosensor green dnd 189, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
8 protocols using recombinant human m csf
1
Macrophage Co-Culture Activation Assay
2
Inflammatory Cytokine Modulation Assay
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LPS from Escherichia coli O111:B4, PHA from Phaseolus vulgaris, H-89 [protein kinase A (PKA) inhibitor], forskolin (adenylate cyclase activator), and PGE2 were obtained from Sigma-Aldrich. AS-605240 [phosphatidylinositol-3-phosphate kinase (PI3K) inhibitor], PF-04418948 (EP2 receptor antagonist), and L-161,962 (EP4 receptor antagonist) were purchased from Cayman Chemical. Ficoll-Hypaque was obtained from GE Healthcare. Recombinant human TGF-β1, recombinant human M-CSF, recombinant human IL-4, and recombinant human GM-CSF were obtained from Miltenyi Biotech.
3
Regulation of Macrophage Activation Pathways
4
Monocyte-derived Macrophages and Dendritic Cells
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Monocytes isolated using Percoll gradient or CD14-magnetic beads (Miltenyi Biotec) were cultured for 5 to 6 d in RPMI 1640 complete medium (Thermo Fisher Scientific) containing 10% fetal calf serum (Biowest), 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mM glutamine (all from Thermo Fisher Scientific). For generation of moDCs, monocytes were cultured in the presence of recombinant human IL-4 (500 U/mL) and GM-CSF (800 U/mL; both from Immunotools). For generation of moMacs, recombinant human M-CSF (50 ng/mL; Miltenyi Biotec) was used. To increase CD169 expression, cells were treated with recombinant human IFNα (1,000 U/mL; Miltenyi Biotec) during the last 2 d of culture. For enrichment of primary DCs, PBMCs were depleted from non-DC populations using biotinylated antibodies against CD3, CD14, CD19, CD56 (10 µg/mL; all produced and validated in-house), and CD16 (5 µg/mL; Biolegend) and streptavidin nanobeads (Biolegend) using an LD Column (Miltenyi).
5
Cord Blood Mononuclear Cell Isolation and Differentiation
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Cord blood was collected into 3.2% sodium citrate and processed within 24 h of collection. Primary CBMCs were isolated from healthy cord blood and cultured at 37°C, 5% CO2, 95% air in complete RPMI‐1640 (containing stable 2.5 mM l ‐glutamine and 0.5 mM sodium pyruvate with 10% FBS). For MDM generation, CBMCs were plated at 2 × 106 cells/ml in complete RPMI‐1640 supplemented with 50 ng/ml recombinant human M‐CSF (Miltenyi) for 7 or 8 days, with 2 supplements of fresh medium and cytokines at days 3 and 5 of culture (Figure S1(A) ). Monocytes were isolated by negative selection using Monocyte Isolation Kit II (Miltenyi). Monocyte purity was assessed using CD14 staining and was routinely >90% (Figure S1(B) ). For moDCs, isolated monocytes were cultured in tissue‐culture dishes at 1 × 106 cells/ml in RPMI 1640 medium containing 10% FBS supplemented with recombinant human IL‐4 (40 ng/ml) and recombinant human GM‐CSF (80 ng/ml) with 1 supplement of fresh medium and cytokines at day 3 of culture. After 6–7 days, immature moDCs (CD14lowDC‐SIGN+), routinely >90% purity, were harvested by gently pipetting only the loosely adherent fraction and replated in a 96‐well format at the desired cell density (Figure S1(C) ). T cells were isolated from CBMCs by negative selection using CD4+ T cell isolation kit (Miltenyi) and were routinely >95% CD45+CD4+ (Figure S1(D) ).
6
Macrophage Differentiation from Murine Bone Marrow and Human PBMCs
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Primary macrophages were differentiated from mouse bone marrow cells or human peripheral blood mononuclear cells (PBMCs). Bone marrow cells were obtained from C57BL/6J male mice 7-12 wks of age and differentiated into mature bone marrow-derived macrophages (BMDMs) for 7 days in RPMI 1640 containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (PS), and 2 mM L-glutamine (GIBCO), supplemented with fresh recombinant murine M-CSF (50 ng/ml; Miltenyi Biotec). The medium was changed on days 3 and 5 with fresh medium containing M-CSF. PBMCs were isolated from healthy donors. Monocytes were separated using magnetic bead selection (Miltenyi Biotec) and cultured in RPMI 1640 containing 10% FBS, 1% PS, and 2 mM L-glutamine, supplemented with fresh recombinant human M-CSF (50 ng/ml; Miltenyi Biotec) every three days for 7 days to differentiate into mature macrophages.
7
Monocyte-Derived Macrophage Expansion Protocol
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CD14+ve monocytes were cultured under low adhesion conditions in six-well plates (Corning) for buffy coat monocytes or MACS Good Manufacturing Practice (GMP) 100 mL expansion bags (Miltenyi Biotec) for all apheresis samples at a density of 2 × 106 cells/mL in the presence of 100 ng/mL human recombinant M-CSF (Miltenyi) in a humidified atmosphere at 37°C (95%O2/5%CO2). Initial cultures were carried out in Iscove's Modified Eagle's Medium supplemented with 10% FBS from a validated GMP-compliant source (Life Technologies Ltd). Further experiments were carried out in serum-free AIMV medium (Invitrogen) to comply with GMP requirements. In all experiments a media change was performed on days 3 and 5. Low-adhesion plates were used because these best mimic the conditions in the expansion bags. Furthermore, if normal tissue culture plastic had been used, the macrophages would readily attach to the plastic, making it difficult to remove them without either mechanical disruption or enzymatic treatment, which could result in increased cell death and an altered macrophage phenotype.
8
Differentiation and Harvesting of Human Monocyte-Derived Macrophages
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CD14+ monocytes were harvested via apheresis from healthy volunteers as described in Moore et al.[12] (link). Monocytes were frozen in liquid nitrogen in cryopreservation solution. For SPION-labeling experiments, high-density monocyte aliquots were defrosted in a water bath, washed, and resuspended in Iscove's Modified Eagle Medium supplemented with 10% fetal bovine serum and 100 ng/mL human recombinant mCSF (Miltenyi) in low-attachment flasks (Corning) at 2 × 106 monocytes/mL for 7 days, replacing mCSF every second day. After 7 days, hMDMs were harvested as described, counted and incubated overnight in 96-well plates (4 × 104/well) before viability studies were performed.
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