Purified monocytes were cultured at a density of 1 × 106 cells per mL in complete medium in the presence of recombinant GM-CSF (5 ng/mL), M-CSF (25 ng/mL), IFN-γ (10 ng/mL), IL-10 (10 ng/mL), and IL-4 (800 U/mL); all from R&D Systems, to generate macrophages. For dendritic cell differentiation, monocytes were cultured in the presence of GM-CSF (800 U/mL) in combination with IL-4 (500 U/mL), or GM-CSF (800 U/mL) in combination with IFN-α2a (1,000 U/mL, Cell Sciences). Monocytes were differentiated for 7 days at 37°C in a 5% CO2 incubator. At day 3, medium was refreshed with the same concentration of recombinant proteins. Cells were harvested at day 7, after 5 min incubation with accutase (Sigma-Aldrich). Next, LAIR-1 expression was determined using flow cytometry, together with the expression of CD14, CD11c, CD163, CD64, CD1a, and CD80, to assess the markers for macrophage and DC differentiation (Supplementary Figure 1 ).
Ifn α2a
Manufactured by Cell Sciences
Sourced in United States
IFN-α2a is a recombinant human interferon alpha-2a protein. It is a cytokine involved in the innate immune response against viral infections.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by R&D Systems (2 mentions)
Tlr4 ligand lps, by InvivoGen (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Gm csf, by Cell Sciences (1 mentions)
Interleukin 4 (il 4), by Cell Sciences (1 mentions)
Accutase, by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
M csf, by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
48 well culture plate, by Corning (1 mentions)
Nunc culture plates, by Thermo Fisher Scientific (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Macs human monocyte isolation kit 2, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
Tgf β2, by Bio-Techne (1 mentions)
4 protocols using ifn α2a
1
Macrophage and Dendritic Cell Differentiation
2
Monocyte-Derived Dendritic Cell (moDC) Generation
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Purified monocytes were cultured in pre-coated 24 well plates, as described above, at a density of 1 × 106 cells per mL in complete medium. To generate moDCs, recombinant human IL-4 (500 U/mL) and GM-CSF (800 U/mL); both from R&D Systems were added to the medium. moDCs were differentiated for 6 days at 37°C in a 5% CO2 incubator and at day 3 medium was supplemented with the same concentrations of IL-4 and GM-CSF. At day 6, cells were either harvest for flow cytometry staining or 100.000 cells were re-seeded in a 48 well culture plate (Corning, Costar) and rested overnight. On the day after, cells were left unstimulated or stimulated with LPS-TLR4 ligand (100 ng/mL, Invivogen) or IFN-α2a (1,000 U/mL, Cell Sciences) for 5 h. Finally, cells were lysed with RLT buffer (Qiagen) and stored at −20°C for further analysis.
3
Anti-LAIR-1 Agonist Stimulation of PBMCs and Monocytes
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24 well Nunc culture plates (Thermo Fisher Scientific) were coated with 10 μg/mL of anti-LAIR-1 agonist (clone Dx26) (6 (link)) or 10 μg/mL of mouse isotype control IgG1 (eBioscience-Thermo Fisher Scientific) diluted in PBS overnight at 4°C. A total of 1 × 106 PBMCs or 0.5 × 106 purified monocytes were seeded in the pre-coated plates with complete medium after incubation with Fc receptor blocking reagent (Miltenyi Biotech). Cells were pre-incubated for 2 h at 37°C in a 5% CO2 incubator and then either left unstimulated or stimulated with LPS-TLR4 ligand (100 ng/mL, Invivogen) or IFN-α2a (1,000 U/mL, Cell Sciences). PBMCs were stimulated overnight at 37°C in a 5% CO2 incubator and then harvested for flow cytometry staining. Monocytes were stimulated for 5 h at 37°C in a 5% CO2 incubator and afterwards supernatants were collected and stored at −80°C and cells were lysed with RLT buffer (Qiagen) and stored at −20°C until further analysis.
4
Isolation and Stimulation of CD14+ Monocytes
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PBMCs were isolated from whole heparinized blood samples from SSc patients and healthy controls or from the buffy coats of healthy controls, by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA). CD14+ monocytes were purified from PBMCs using the MACS Human Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) on the autoMACs Pro Separator (Miltenyi Biotec) according to the manufacturer’s instructions. For subsequent analysis, only cell preparations with more than 95% purity (measured by FACS analysis) for CD14+ cells were used.
For selected experiments, CD14+ monocytes purified from buffy coats were cultured in RPMI 1640 + 10% FCS (fetal calf serum, <0.5 EU/mL, Sigma-Aldrich, St. Louis, MO, USA) + 2 mM Glutamine at a concentration of 2 × 106 cells/mL. Cultured cells were left untreated (medium control) or treated with one of the following stimuli: 100 ng/mL ultra-pure E. coli lipopolysaccharide (LPS, strain O111:B4, Invivogen, San Diego, CA, USA), 5 µM R848 (Invivogen), 1000 U/mL IFNα-2a (Cell Sciences, Newburyport, MA, USA), and TGF-β2 (Bio-Techne, Minneapolis, MN, USA) according to the conditions and times indicated for each experiment in the results section.
For selected experiments, CD14+ monocytes purified from buffy coats were cultured in RPMI 1640 + 10% FCS (fetal calf serum, <0.5 EU/mL, Sigma-Aldrich, St. Louis, MO, USA) + 2 mM Glutamine at a concentration of 2 × 106 cells/mL. Cultured cells were left untreated (medium control) or treated with one of the following stimuli: 100 ng/mL ultra-pure E. coli lipopolysaccharide (LPS, strain O111:B4, Invivogen, San Diego, CA, USA), 5 µM R848 (Invivogen), 1000 U/mL IFNα-2a (Cell Sciences, Newburyport, MA, USA), and TGF-β2 (Bio-Techne, Minneapolis, MN, USA) according to the conditions and times indicated for each experiment in the results section.
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