Pigtailed macaque peripheral blood mononuclear cells (PBMCs) were cultured in macrophage differentiation media (MDM) containing DMEM supplemented with 20% human AB serum (Gemini Bio Products), 50 ng/mL recombinant human macrophage colony-stimulating factor (R&D Systems), 2 mM sodium pyruvate (Sigma), 2 mM l -glutamine (Life Technologies), 1 mM HEPES buffer (Life Technologies), and 20 µg/mL gentamicin (Gibco) for 7 days until mature macrophages were visible. Macrophages were then washed 3× with PBS and switched to MDM with 10% human AB serum. Twenty-five nanograms per milliliter of rhesus IFNγ (R&D Systems) or 400 U/mL human IFNβ1a (PBL Interferon Source) were added in duplicate wells for each time point. After 0, 24, 48, and 72 h, supernatants were saved for KP metabolite analysis, and cells were washed and then harvested with gentle scraping for KP metabolite and IDO1 gene expression analysis. Metabolites were normalized to controls at each time point as well as to tyrosine (TYR), which does not change during HIV infection (18 (link)), to control for minor variations in cell number that could affect metabolism.
Recombinant human macrophage colony stimulating factor
Manufactured by R&D Systems
Sourced in United States
Recombinant human macrophage colony-stimulating factor (rhM-CSF) is a protein that stimulates the production and differentiation of macrophages from bone marrow precursor cells. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Human ab serum, by Gemini Bio (1 mentions)
Minimum essential medium (mem), by Corning (1 mentions)
Hepg2 human hepatocellular carcinoma cells, by American Type Culture Collection (1 mentions)
Macs cd14 microbeads, by Miltenyi Biotec (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Ficoll hypaque, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by R&D Systems (1 mentions)
Cd14 micro magnetic beads, by Miltenyi Biotec (1 mentions)
Atcc pcs 100 012, by American Type Culture Collection (1 mentions)
11 protocols using recombinant human macrophage colony stimulating factor
1
Macrophage Differentiation and Kynurenine Pathway
2
Cultivation and Infection of HepG2 Cells and Primary Monocytes
3
Generating Macrophages from Human Monocytes
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Human monocyte-derived macrophages (HMDMs) were prepared as previously described [48 (link)]. CD14-positive monocytes were separated with MACS CD14 micro beads (Miltenyi Biotec) according to the manufacturer’s instructions. Purified monocytes were plated in 96-well plates and differentiated into macrophages by adding 100 ng/mL of recombinant human macrophage colony-stimulating factor (R&D Systems) into RPMI 1640 medium with 10% fetal bovine serum. HMDMs were allowed to attach for at least 3 hours, and then incubated with either 100 ng/mL of LPS or 20 ng/μL of NETs for 24 hours. Supernatant was collected and applied in ELISAs according to the manufacturer’s protocol. ELISA kits of IL-1β, IL-6 and TNF-α were purchased from Invitrogen.
4
Monocyte Isolation and Differentiation
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Purification of monocytes from peripheral blood was performed using Ficoll-Paque (GE Healthcare, Sweden) density gradient centrifugation and immunomagnetic positive selection of CD14+ monocytes using MS columns and CD14 microbeads (Miltenyi Biotec, UK) The % median [IQR] purity was 97 [88–97]% as assessed by flow cytometry using CD14-Alexa Fluor®647 conjugated antibody (Biolegend, London, UK) on a Becton Dickinson FACS Canto II. The monocytes were incubated for 6 days at 37°C and 5% CO2 with Dulbecco modified Eagle medium (DMEM) with high glucose, L-glutamine, D-glucose, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), without sodium pyruvate, phenol red (Fisher Scientific, UK) and supplemented with 10% foetal calf serum, 1% Non-essential amino acids, 1% mixture of antibiotics and antifungal (10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin), and 25 μg/mL of amphotericin B) and 1% Sodium pyruvate) in the presence of recombinant human macrophage colony-stimulating factor (100 ng/ml) (R&D Systems, Europe).
5
Isolation and Differentiation of Human Monocyte-Derived Macrophages
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Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood through density gradient centrifugation using Ficoll. In brief, blood collected into EDTA-coated tubes was diluted 1:1 with sterile PBS and layered on Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ), followed by centrifugation for 30 min at 500g without applying a brake. The PBMCs in the interface were carefully removed and washed twice with PBS. PBMCs were then placed in a 6-well plate for 2 h, and then adherent cells (monocytes) were cultured in RPMI-1640 medium supplemented with 10% FBS and 10 ng/mL recombinant human macrophage colony-stimulating factor (R&D, Minneapolis, MN) for 4 days. Media were replaced once at day 2.
6
Human Monocyte-Derived Macrophage Isolation
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Human monocyte-derived macrophages were obtained under research protocol (UCSD IRB-12-0902) approved by the University of California, San Diego Institutional Review Board. Following informed consent, de-identified blood was obtained from patients undergoing cardiac catheterization from the arterial sheath after access achieved but prior to any administration of contrast agent. Human macrophages were prepared from CD14+ peripheral blood mononuclear cells (PBMCs) isolated from human blood by incubating in RPMI-1640 + 10% FBS supplemented with penicillin/streptomycin and 50 ng/mL recombinant human macrophage colony-stimulating factor (R&D Systems) (refer to Supporting Information for additional details).
7
Isolation and Differentiation of Human Monocytes
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Peripheral blood was obtained from healthy donors after informed consent. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Life Technologies). Monocytes were purified by magnetic-associated cell sorting by negative selection. Differentiation used 50 ng ml−1 of recombinant human macrophage colony-stimulating factor (R&D systems) in cRPMI.
8
Isolation and Differentiation of Monocytes
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Peripheral blood mononuclear cells (PBMCs) were prepared on a Ficoll‐Hypaque density gradient centrifugation. CD14+ cells were obtained through positive selection by CD14+ micromagnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, monocytes were differentiated in complete RPMI‐1640 medium supplemented with recombinant human macrophage colony‐stimulating factor (50 ng/mL; R&D Systems, Minneapolis, MN) for an additional 6 days before following study. Human primary aortic smooth muscle cells (HASMCs; ATCC PCS‐100‐012; American Type Culture Collection [ATCC], Manassas, VA) and human primary aortic endothelial cells (HAECs; ATCC PCS‐100‐011; ATCC) were cultured according to the manufacturer's instructions. Cells were used for experiments at passages 3 to 8.
9
Isolation and Differentiation of Human Macrophages
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We isolated human monocytes from PBMCs by using a CD14-positive selection system (MACS system, Miltenyi Biotec Inc.), followed by cultivation in RPMI-1640 medium supplemented with 10% FBS, 50 mM 2-mercaptoethanol (Sigma-Aldrich, USA), and 50 ng/ml recombinant human macrophage colony-stimulating factor (R&D systems, USA) for 5 days to differentiate into macrophages. The culture medium was supplemented on the 3rd day of differentiation. This process was also described previously [12 (link)].
10
Isolation and Differentiation of Human Macrophages
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Blood of healthy donors was used (Etablissement Français du Sang, Ile de France, Site Trinité, France) with the appropriate prior ethics approval as stated in the Etablissement Français du Sang and INSERM agreements 15/EFS/012 and 18/EFS/030, ensuring that all donors gave a written informed consent and providing anonymized samples. Primary human peripheral blood mononuclear cells were isolated by density-gradient sedimentation in Ficoll (GE Healthcare, Waukesha, WI, USA). Then, monocytes were selected by adhesion to dishes for 2 h at 37°C in fetal calf serum (FCS)–free medium [Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 100 U/ml penicillin 100 μg/ml streptomycin and 2 mM l -glutamine (Thermo Fisher Scientific)]. They were differentiated into macrophages for 8 d in adhesion medium supplemented with 10% FCS and 10 ng/ml recombinant human macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA) as in (22 (link), 23 (link)).
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