Monocytes were isolated from heparinized peripheral blood via negative selection (RosetteSep, StemCell) followed by density gradient centrifugation over Ficoll separation medium (GE). Monocytes were maintained at 106/mL in DMEM (Gibco) with 10% heat-inactivated human serum and 50 ng/mL each of recombinant human GM-CSF and IL-4 (Peprotech) in ultra-low attachment plates (Corning) for 7-10 days. Small MPs (containing VD3 or equivalent mass unloaded PLGA, 5 µg) were incubated in wells with harvested DC in ultra-low attachment plates to allow for antigen/PLGA uptake. Large MPs (containing TGF-β1 and GM-CSF or equivalent mass unloaded PLGA) were added at 5 mg per 106 DC in 0.4 μM pore size hanging well inserts (Miltenyi) to prevent cell overcrowding, for two days prior to testing for phenotype or stimulus response.
Recombinant human gm csf and il 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant human GM-CSF and IL-4 are cytokines produced through recombinant DNA technology. GM-CSF stimulates the production and function of granulocytes and macrophages, while IL-4 promotes the differentiation of naive T cells into Th2 cells.
Automatically generated - may contain errors
Lab products found in correlation
Aim 5 medium, by Thermo Fisher Scientific (2 mentions)
Rosettesep human monocytes enrichment cocktail, by STEMCELL (2 mentions)
Cgmp grade mpla, by Avanti Polar Lipids (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Aim 5 culture medium, by Thermo Fisher Scientific (1 mentions)
Rosettesep human monocyte enrichment cocktail, by STEMCELL (1 mentions)
5 protocols using recombinant human gm csf and il 4
1
Generation of Monocyte-Derived Dendritic Cells
2
Dendritic Cell Differentiation and Antigen Presentation
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DCs were differentiated in vitro from the peripheral blood adherent mononuclear cells (PBMCs) of six healthy donors. The PBMCs were obtained by centrifugation through a Ficoll-isopaque gradient, suspended in AIM-V culture medium (Invitrogen), and seeded in 6-well culture plates (1x106/per well). After incubating for 1 h at 37˚C, non-adherent cells were removed and adherent monocytes were cultured in complete culture medium containing 80 ng/ml recombinant human GM-CSF and IL-4 (PeproTech) for 5 days. They were then treated with HCT-116 lysate (100 µg per 106 DCs) and kept in culture for an additional 48 h. Immature DCs were submitted to seven different culture conditions: DCs (untreated immature DCs); WT (DCs exposed to the lysate of untreated HCT-116 cells); CQ (DCs exposed to the lysate of HCT-116 cells that were pretreated with 80 µM of CQ), 5-FU (DCs exposed to the lysate of HCT-116 cells that were pretreated with 20 µM of 5-FU), and 5-FU + CQ (DCs exposed to the lysate of HCT-116 cells that were pretreated with 20 µM of 5-FU and 80 µM of CQ). All procedures involving both normal and transformed human cells were approved by the Ethics Committee of the Botucatu School of Medicine – UNESP (CEP # 2.258.145).
3
Generation and Characterization of Dex-Modulated DCs
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Human moDCs were generated from monocytes as previously described (20 (link)). Monocytes were isolated from peripheral blood of 10 healthy individuals by negative selection using RosetteSep Human Monocytes enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. Monocytes were cultured at 2 × 106 cells/ml in serum-free AIM-V medium (Gibco BLR, Grand Island, NY, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) for 5 days at 37°C and 5% CO2. At day 3, culture medium was replenished and cells were incubated with Dex (Sigma-Aldrich, St. Louis, CO, USA) at a final concentration of 1 µM [Dex-modulated DCs (D-DCs)]. At day 4, cells were stimulated with 1 µg/ml of cGMP-grade MPLA (Avanti Polar Lipids Inc., Alabaster, AL, USA) (DM-DCs). Unstimulated cells (DCs) and MPLA-matured DCs (M-DCs) generated in the absence of Dex were used as controls of immature and mature DCs, respectively. On day 5, cells were harvested and characterized by flow cytometry.
4
Generation and Characterization of Human Monocyte-Derived Dendritic Cells
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Human moDCs were generated from monocytes as previously described (10 (link)). Monocytes were isolated from peripheral blood by negative selection using RosetteSep Human Monocytes enrichment cocktail (Stemcell Technologies, Vancouver, Canada) according to manufacturer's instructions. Monocytes were cultured at 2 × 106 cells/ml in serum-free AIM-V medium (Gibco BLR, Grand Island, NY, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) for 5 days at 37°C and 5% CO2. At day 3, culture medium was replenished and cells were incubated with dexamethasone (Sigma-Aldrich, St. Louis, CO, USA) at a final concentration of 1 μM. At day 4, cells were stimulated with 1 μg/ml of cGMP-grade MPLA (Avanti Polar Lipids Inc., Alabaster, AL, USA) (DM-DCs). Unstimulated cells (DCs) and MPLA-matured DCs (M-DCs) generated in the absence of dexamethasone were used as controls of immature and mature DCs, respectively. On day 5, cells were harvested and characterized by flow cytometry. Monocyte purity and gating strategy for DC characterization are shown on Supplementary Figure S1 .
5
Generation of Modulated Dendritic Cells
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Monocytes were isolated by negative selection using RosetteSep Human Monocyte enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. moDCs were generated as previously described (20 (link)) in AIM-V medium (Gibco BLR, Grand Island, NE, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) within 5 days. At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA). Untreated/immature DCs (iDCs) and MPLA-matured DCs (mDCs) were used as controls.
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