Whole blood (~8 mL) was collected from consenting healthy donors using Vacutainer CPT Mononuclear Cell Preparation Tubes (CPT; BD) containing sodium citrate. Tubes were processed according to the manufacturer’s instructions to separate peripheral blood mononuclear cells (PBMCs). PBMCs were washed twice in PBS (5 min at 500 × g) at room temperature before being resuspended in complete media (RPMI, 10%FBS, 1% penicillin-streptomycin, and 10 ng mL−1 recombinant human macrophage colony stimulating factor (M-CSF; [R&D Systems]). Isolated PBMCs (1x106 mL−1) were plated on 12 well tissue culture-treated plates in complete media. Complete media was refreshed every 48 hours. After 7 days, adherent M-CSF-derived cells were treated with 100 ng mL−1 lipopolysaccharide from E. coli 055:B5 (LPS; Sigma) and 5 ng mL−1 recombinant human interferon gamma (IFN-γ; R&D Systems) to induce M1 macrophage polarization or with 20 ng mL−1 recombinant human interleukin 4 (IL-4; R&D Systems) to induce M2 macrophage polarization. Cells were harvested and RNA extracted 24 hrs later.
Recombinant human interleukin 4 il 4
Manufactured by R&D Systems
Sourced in United Kingdom, United States
Recombinant human interleukin-4 (IL-4) is a cytokine that plays a key role in the immune response. It is involved in the differentiation of naive helper T cells into Th2 cells.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human macrophage colony stimulating factor m csf, by R&D Systems (1 mentions)
M csf, by Merck Group (1 mentions)
Recombinant human interferon gamma ifn γ, by R&D Systems (1 mentions)
Rpmi media, by Thermo Fisher Scientific (1 mentions)
Rpmi media, by R&D Systems (1 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Gm csf, by R&D Systems (1 mentions)
L glutamate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
5 protocols using recombinant human interleukin 4 il 4
1
PBMC Isolation and Macrophage Polarization
2
Isolation and Polarization of Human Macrophages
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The preparation of human monocytes from buffy coats of healthy volunteers was performed using Ficoll-Hypaque (Pharmacia Corporation) for 50 min at 400 g. Twenty-four-well plates were seeded with 2×10 6 cells/mL in RPMI 1640 medium containing 10% heat inactivated human AB serum, 50 U of penicillin/mL, 50 U of streptomycin/mL, 2 mM L-glutamine, and 100 ng/mL human M-CSF (which allows differentiation into macrophages). Warm medium was used to gently wash away non-adherent cells 6 days post-culture. CD14 + macrophages were found to account for greater than 95% of the adherent cells. The activation of these monocytes to macrophages in vitro involved the treatment of 2×10 6 cells/L with 25 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) to produce M1-polarized macrophages and 45 ng/mL recombinant human interleukin-4 (IL-4; R&D) to produce M2-polarized macrophages. Flow cytometry was employed to detect the formation of macrophages. For the following in vitro assays, cells were cultured for 24 hours with RPMI media minus supplements and meticulously washed with PBS prior to the experiments.
3
Isolation and Polarization of Human Macrophages
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The preparation of human monocytes from buffy coats of healthy volunteers was performed using Ficoll-Hypaque (Pharmacia Corporation) for 50 min at 400 g. Twenty-four-well plates were seeded with 2×10 6 cells/mL in RPMI 1640 medium containing 10% heat inactivated human AB serum, 50 U of penicillin/mL, 50 U of streptomycin/mL, 2 mM L-glutamine, and 100 ng/mL human M-CSF (which allows differentiation into macrophages). Warm medium was used to gently wash away non-adherent cells 6 days post-culture. CD14 + macrophages were found to account for greater than 95% of the adherent cells. The activation of these monocytes to macrophages in vitro involved the treatment of 2×10 6 cells/L with 25 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) to produce M1-polarized macrophages and 45 ng/mL recombinant human interleukin-4 (IL-4; R&D) to produce M2-polarized macrophages. Flow cytometry was employed to detect the formation of macrophages. For the following in vitro assays, cells were cultured for 24 hours with RPMI media minus supplements and meticulously washed with PBS prior to the experiments.
4
Isolation and Differentiation of Monocytes and Macrophages
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Monocytes were isolated using a Pan Monocyte Isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions or by adherence for 2 hr in RPMI media containing no serum. Macrophages were differentiated from monocytes cultured under adherence conditions for 6 days in RPMI media (Gibco, UK) supplemented with 50 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D, UK). MDDCs were obtained from monocytes cultured under adherence conditions for 6 days in RPMI media supplemented with 50 ng/ml of human recombinant interleukin-4 (IL-4; R&D, UK) and 50 ng/ml GM-CSF.
5
Monocyte-Derived Dendritic Cell Generation
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DC were generated from peripheral blood monocytes. Briefly, PBMCs were washed three times with PBS (Sigma Aldrich, USA) and resuspended at the concentration of 3 106 cell/mL in RPMI-1640 medium (Sigma Aldrich, USA) plus 1% FBS (Gibco, UK) and 2 mM L-glutamate (Gibco, UK) at 37 °C in a humidified CO2 incubator. After 2 h, non-adherent cells were removed by rinsing with PBS and human recombinant interleukin-4 (IL-4) (R&D Systems) at a final concentration of 1000 U/mL and human recombinant granulocyte-monocyte colony-stimulating factor (GM-CSF) at a final concentration of 800 U/mL (R&D Systems) were added After 5 days of incubation, monocyte-derived DCs (mo-DC) were matured with the addition of lipopolysaccharide (LPS) (Sigma Aldrich, USA) for 1 additional day. Mo-DCs were pulsed with BKPyV capsid protein VP1, and BKPyV-LargeT antigen PepMixes (JPT Peptide Technologies). PepMixes are overlapping 15-mer peptide mixtures that cover the entire length of both antigens.
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