Human monocyte-derived DCs were generated as previously described31 (link). In brief, human peripheral blood mononuclear cells (PBMCs) were harvested by density gradient centrifugation using Biocoll (Biochrom AG). CD14+ primary human monocytes were further purified from PBMCs by CD14+ microbeads (Miltenyi Biotech) using a magnetic cell sorting system. Primary monocytes were differentiated into monocyte-derived dendritic cells (moDCs) by treatment with 800 U/ml human recombinant GM-CSF and 1,000 U/ml human recombinant IL-4 (Miltenyi Biotech). At day six cell surface marker expression (Fig. S1A ) and phenotype of immature moDCs was monitored prior to performing conidia interaction studies.
Recombinant human il 4
Manufactured by Miltenyi Biotec
Recombinant human IL-4 is a cytokine produced using recombinant DNA technology. It functions as a regulator of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human gm csf, by Miltenyi Biotec (3 mentions)
Cd14 microbead, by Miltenyi Biotec (3 mentions)
Biocoll, by Harvard Bioscience (1 mentions)
Iscove modified dulbecco media (imdm), by PAN Biotech (1 mentions)
Recombinant human m csf, by Miltenyi Biotec (1 mentions)
Forskolin, by Merck Group (1 mentions)
Pha from phaseolus vulgaris, by Merck Group (1 mentions)
As 605240, by Cayman Chemical (1 mentions)
Pf 04418948, by Cayman Chemical (1 mentions)
Recombinant human tgf β1, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Anti cd14 conjugated magnetic microbeads, by Miltenyi Biotec (1 mentions)
Rpmi 1640, by Miltenyi Biotec (1 mentions)
Lymphocyte separation media, by Biosera (1 mentions)
Lymphoprep, by Cedarlane (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
9 protocols using recombinant human il 4
1
Generation of Monocyte-Derived Dendritic Cells
2
Investigating STAT6 Variants in Lymphoma Cells
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OCI-Ly1and OCI-Ly8 cells were cultured in IMDM (PAN Biotech, Aidenbach, Germany). 293 T HEK cells and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium. All cells were cultured with 10% FBS (PAN), 37 °C 5% CO2. Cell lines were authenticated by short tandem repeat analysis (Eurofins, Val Fleuri, Luxembourg) and tested negative for mycoplasma by PCR. Of note, we confirmed that OCI-Ly1 harbors a variant (G375R) in the STAT6 DNA-binding domain [23 (link)] that, to the best of our knowledge, has not been reported in any other cell line or primary tumor sample. Cells were stably transduced with a CMV-driven cDNA expression construct (pHAGE-CMV-MCS-IRES-ZsGreen; PlasmID, EvNO00061605) encoding for Flag-tagged mutant (MUT) STAT6 (D419G, D419N, N421K, or D519V) or wild type (WT) STAT6 (PlasmID, HsCD00365550) as previously described [7 (link)], and stimulated with human recombinant IL-4 (Miltenyi Biotec, Cologne, Germany) as indicated.
3
Inflammatory Cytokine Modulation Assay
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LPS from Escherichia coli O111:B4, PHA from Phaseolus vulgaris, H-89 [protein kinase A (PKA) inhibitor], forskolin (adenylate cyclase activator), and PGE2 were obtained from Sigma-Aldrich. AS-605240 [phosphatidylinositol-3-phosphate kinase (PI3K) inhibitor], PF-04418948 (EP2 receptor antagonist), and L-161,962 (EP4 receptor antagonist) were purchased from Cayman Chemical. Ficoll-Hypaque was obtained from GE Healthcare. Recombinant human TGF-β1, recombinant human M-CSF, recombinant human IL-4, and recombinant human GM-CSF were obtained from Miltenyi Biotech.
4
Isolation and Culture of Dendritic Cells
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Peripheral blood samples were obtained from healthy donors. Informed consent was provided according to the Declaration of Helsinki. The PBMCs were isolated by density gradient centrifugation while using Lymphocyte Separation Media (Biosera, Monza, Italy). The DCs were prepared, as previously described [29 (link)], with minor modifications. Briefly, monocyte purification was obtained by positive selection, while using anti-CD14 conjugated magnetic microbeads (Miltenyi Biotec, Bologna, Italy). Cell purity, as assessed by flow cytometry (FACS), was always ≥ 95%. Freshly purified monocytes were cultured for five days at the density of 350,000 cells/ml in complete medium (RPMI 1640, 10% endotoxin-free FBS, 2 mM L-Glutamine, and 1% Penicillin-Streptomycin) supplemented with 20 ng/mL of recombinant human IL-4 and 50 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) both purchased from Miltenyi Biotec to obtain immature DCs (immDCs).
5
Isolation and Differentiation of Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy peripheral blood by density gradient centrifugation over Lymphoprep (CEDARLANE) and washed with PBS. Red blood cells were lysed by incubation with ammonium chloride solution and platelets were removed by low-speed centrifugation.
CD14+ monocytes were isolated from PBMCs by positive selection using CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.
CD14+ monocytes were differentiated into dendritic cells (DC) in RPMI 1640 medium (Lonza), 10% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin/streptomycin (Lonza), and 2 mM L-glutamine (Lonza) at 106 cells/mL, in the presence of 100 ng/mL Recombinant Human GM-CSF (Miltenyi Biotec) and 10 ng/mL Recombinant Human IL-4 (Miltenyi Biotec) for 7 days at 37°C with 5% CO2. Fresh medium with cytokines, at concentrations stated above, was added on day 3. Mature DC were obtained by adding 1 mg/mL lipopolysaccharide at day 5 of differentiation.
DC-10 were generated as previously described.17 (link) Briefly, CD14+ monocytes were cultured as described above for DC differentiation, with the addition of Recombinant Human IL-10 (CellGenix) at 10 ng/mL.
CD14+ monocytes were isolated from PBMCs by positive selection using CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.
CD14+ monocytes were differentiated into dendritic cells (DC) in RPMI 1640 medium (Lonza), 10% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin/streptomycin (Lonza), and 2 mM L-glutamine (Lonza) at 106 cells/mL, in the presence of 100 ng/mL Recombinant Human GM-CSF (Miltenyi Biotec) and 10 ng/mL Recombinant Human IL-4 (Miltenyi Biotec) for 7 days at 37°C with 5% CO2. Fresh medium with cytokines, at concentrations stated above, was added on day 3. Mature DC were obtained by adding 1 mg/mL lipopolysaccharide at day 5 of differentiation.
DC-10 were generated as previously described.17 (link) Briefly, CD14+ monocytes were cultured as described above for DC differentiation, with the addition of Recombinant Human IL-10 (CellGenix) at 10 ng/mL.
6
Regulation of B Cell Viability by CD57+ T Cells
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Live blood CD57+ CD4+ T cells (7AAD− CD19− CD3+ CD8− CD57+), CD57+ CD4+ T cells (7AAD− CD19− CD3+ CD8− CD57−) and B cells (7AAD− CD19+ CD3−) were purified from fresh PBMCs of healthy donors by flow cytometry. B cells were incubated with CD57+ or CD57− CD4+ T cells for 24 h (10 T: 1 B) in the presence or absence of SEB (200 ng/ml) with recombinant human IL-4 (20 ng/ml; Miltenyi Biotec, 130-093-919) and IL-21 (20 ng/ml; Miltenyi Biotec, 130-095-784), followed by Annexin V and 7AAD analysis.
7
Purification of Human Dendritic Cells
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Endotoxin-free reagents and plastic materials were used in all experiments. RPMI-1640, phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Invitrogen, Argentina). Twenty-four-well flat bottom polystyrene plates were purchased from Jet-biofil (AP Biotech, Buenos Aires, Argentina) while 96-well U-bottom plates and half-area 96-well ELISA were obtained from Greiner Bio One (GBO, Buenos Aires, Argentina). Ficoll-Paque PLUS and Percoll were obtained from GE Healthcare Life Sciences (Embiotec, Buenos Aires, Argentina). Recombinant human IL-4 and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Miltenyi Biotec (Lab Systems, Buenos Aires, Argentina). Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma-Aldrich (Merck, Argentina).
8
Expansion and Cryopreservation of B Cells
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PBMC were thawed and resuspended in prewarmed Iscove’s Modified Dulbecco Medium (IMDM, Gibco) with 10% heat-inactivated human AB serum (Innovative Research, lot pretested for performance for B-cell expansions), penicillin-streptomycin, L-glutamine (both from Sigma), and insulin-transferrin-selenium (Thermo Fisher Scientific), recombinant human multimeric CD40L (Human CD40-ligand multimer kit, Miltenyi Biotec), recombinant human IL-4 (Miltenyi Biotec) and recombinant human IL-21 (ImmunoTools) (B-cell medium), supplemented with cyclosporine A (Novartis). On days 6–8, B cells were enriched using CD19 MicroBeads (Miltenyi Biotec) and resuspended in prewarmed B-cell medium. Afterwards, B cells were counted every 72–96 hours and resuspended in prewarmed B-cell medium. When B-cell numbers were sufficient to perform T-cell assays, B cells were frozen in Cryostor CS10 medium (StemCell Technologies) prior to their use as antigen-presenting cells (APCs) in T-cell assays.
9
Isolation and Culture of Myeloid Cells
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PBMCs were isolated from heparinized peripheral blood from healthy donors by density gradient centrifugation on Lymphoprep (Nycomed). For DC, macrophage, or LC culture, monocytes were isolated using Percoll (Pharmacia) density gradients. Cells were cultured for 6 days in IMDM (Lonza) containing 5% fetal bovine serum (FBS; Biowest) and 86 μg/mL gentamicin (Gibco) supplemented with 20 ng/mL recombinant human GM‐CSF (Invitrogen) and 2 ng/mL recombinant human IL‐4 (Miltenyi Biotec) for DCs, 20 ng/mL recombinant human GM‐CSF for macrophages, or 20 ng/mL recombinant human GM‐CSF, 2 ng/mL recombinant human IL‐4 and 10 μg/mL recombinant human TGF‐β1 (R&D Systems) for LCs. At day 2 or 3, half of the medium was replaced by fresh medium containing cytokines. Monocytes were isolated from PBMCs by MACS isolation using CD14 microbeads (Miltenyi Biotec) for direct stimulation or analysis.
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