Human PBMCs from healthy donors were obtained immediately after blood withdrawal using the Ficoll-Paque (GE Healthcare) gradient method and stored in liquid nitrogen until usage. Cells were thawed in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (Access Biologicals) and 1% penicillin/streptomycin (Gibco). CD14+ CD16+ monocytes were then enriched from total PBMCs by negative selection using the EasySepTM human monocyte enrichment kit without CD16 depletion (STEMCELL Technologies) according to the manufacturer's protocol and counted. Cells were then resuspended in serum-free CellGenix GMP dendritic cell medium (CellGenix) with 100 ng/ml of recombinant human GM-CSF (BioLegend) and 20 ng/ml of recombinant human IL-4 (Gemini Bio-Products) at a density of 2 × 106 per ml in 24-well plates. After 48 h of incubation, cells were stimulated with 0.5 ug/ml of LPS (Invivogen) plus 40 ng/ml of IFNγ (Gemini Bio-Products) in medium or with two concentrations 25 and 50uM of the STING agonist MO4 (source) in DMSO, and compared to the DMSO control. Dendritic cells were harvested after 24 h of stimulation, and analyzed by flow cytometry.
Recombinant human gm csf
Manufactured by BioLegend
Sourced in United States
Recombinant human GM-CSF is a cytokine that stimulates the production and function of granulocytes and macrophages. It is a glycoprotein that can be used for in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Interleukin 4 (il 4), by BioLegend (3 mentions)
Rpmi 1640 glutamax culture medium, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Easysep human monocyte enrichment kit without cd16 depletion, by STEMCELL (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Lipopolysaccharides (lps), by InvivoGen (2 mentions)
Gmp dendritic cell medium, by CellGenix (2 mentions)
Recombinant human il 4, by Gemini Bio (2 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Anti human cd14 beads, by Miltenyi Biotec (1 mentions)
Recombinant human il 4, by BioLegend (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Cd14 cells, by Miltenyi Biotec (1 mentions)
Ada 1, by Merck Group (1 mentions)
8 protocols using recombinant human gm csf
1
Monocyte-Derived Dendritic Cell Activation
2
Protocol for Human Dendritic Cell Differentiation and Maturation
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Human DCs were differentiated as described elsewhere.65 (link) Briefly, magnetic sorted CD14+ cells (Sorted with CD14 Microbeads Human, MACS Miltenyi; #130-050-201) from PBMCs of healthy donors were cultured in 75 cm2 cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (Invitrogen). For DCs differentiation, on day 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF (#572904; BioLegend) and 50 ng/mL IL-4 (#574006; BioLegend). The cultures were maintained at 37°C in humidified atmosphere with 5% CO2. For DCs maturation, on day 5 of culture, LPS (#L2630-10 MG; Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued for another 48 h. The cells were harvested and used for phenotyping through staining for the surface markers CLIP (clone CerCLIP.1; #ab22606; Abcam, Cambridge, UK), MHC-II (clone Tü39; #555556; BD), CD80 (#557226; BD), CD83 (#556855; BD) and CD86 (#555660; BD) or in co-cultures with T-cells.
3
Monocyte-derived DCs Activation by HGSOC IgGs
4
Generating Mature Dendritic Cells from Monocytes
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Human DCs were differentiated from monocytic CD14+ precursors as described previously (37 (link)). Briefly, magnetic sorted CD14+ cells from PBMCs of healthy donors were cultured in 175 cm2 cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen). For DC differentiation, on day 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF and 50 ng/mL IL-4 (BioLegend). The cultures were maintained at 37°C in humidized atmosphere with 5% CO2. For DC maturation, on day 5 of culture, LPS (Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued. The cells were harvested on day 7 and used in T cell stimulation assays.
5
Dendritic Cell Differentiation and Maturation
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DCs were generated in vitro from magnetic-sorted CD14+cells (Miltenyi Biotec, Bergisch Gladbach, Germany) from PBMCs of healthy donors. Briefly, CD14+cells were cultured in 175 cm2 cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, California, USA) with 10% heat-inactivated fetal bovine serum (Gibco), 1% pen/strep (Thermo Scientific), 1% sodium pyruvate (Thermo Scientific) and 1% non-essential amino acids (Thermo Scientific). For differentiation of CD14+cells into immature DCs (iDCs), on days 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF and 50 ng/mL IL-4 (BioLegend, San Diego, California, USA). The cultures were maintained at 37°C in humidized atmosphere with 5% CO2. For DCs maturation, on day 5 of culture, LPS (Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued. The cells were harvested on day 7, loaded with peptides and used in co-cultures with autologous CD8+T cells.
6
Monocyte-Derived Dendritic Cell Activation
7
Isolation and Culture of Diverse Human Cell Types
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HEK293T and THP-1 cells were obtained from ATCC and cultured in DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1 U/ml penicillin and 100 mg/ml streptomycin (Gibco). Human CD14 + cells were isolated from healthy donor PBMCs using MojoSort Human CD14 Selection Kit (Biolegend #480026), and were cultured with 50 ng/ml recombinant human GM-CSF (BioLegend #572904) and 50 ng/ml recombinant human M-CSF (BioLegend #574806) for 6 days to obtain monocytes derived macrophages. CD4 + T cells from human tonsil biopsies were purified by sorting. Blood CD4 + T cells were isolated from healthy donor or patient PBMCs using a human CD4 + T cell isolation kit (BioLegend #480010). Purified CD4 + T cells were used without stimulation, or co-stimulated with plate-bound CD3 (Biolegend #300333) and soluble CD28 (Biolegend #302943) antibodies with the presence of 20 ng/μl IL-2 (Biolegend #589106) for 3 days.
8
Isolation and Differentiation of Human Monocyte-Derived Macrophages
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Buffy coats from healthy donors (n = 2) were obtained from the Australian Red Cross blood services (Brisbane, Australia), and the sex and age of the donors are anonymous. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, UK). Enriched monocyte population was obtained by positive selection using CD14 + magnetic microbeads (Miltenyi Biotec, Germany) . Macrophage differentiation was performed as previously described (Tarique et al., 2015) . Briefly, monocytes were cultured in RPMI-1640 (Lonza, USA), supplemented with 10% heat inactivated fetal bovine serum (FBS) (Life Tech, USA), 1% penicillin-streptomycin-Fungizone (Lonza, USA), and recombinant human GM-CSF (50 ng/mL; BioLegend, USA) for 6 days. Medium was refreshed with GM-CSF (50 ng/mL) on day 3. Human Ethics application was approved by Children's Health Queensland and the approval number is HREC/16/QRCH/379.
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