B cells were isolated by negative magnetic bead selection using the MACS B Cell Isolation Kit II (Miltenyi Biotec) according to manufacturer’s instructions. The purity of >95% was reached in B cell fractions. The cells were stimulated in vitro for 9 days at 37°C in RPMI 1640 medium containing 10% FCS either in the presence of IL4, IL21, CD40L or a combination of IL4+ CD40L +/− IL21. IL4 (ImmunoTools) was used at the final concentration of 100 U/ml. Preparation of CD40L and IL21 was previously described [18] . Prior to use CD40L and IL21 containing supernatants were concentrated and titrated.
Interleukin 4 (il 4)
Manufactured by Immunotools
Sourced in Germany, United States
IL-4 is a recombinant human cytokine. It is involved in the regulation of immune responses and cellular differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by Immunotools (23 mentions)
Ifn γ, by Immunotools (16 mentions)
Lipopolysaccharides (lps), by Merck Group (14 mentions)
Il 13, by Immunotools (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Interleukin 6 (il 6), by Immunotools (3 mentions)
Il 1β, by Immunotools (3 mentions)
M csf, by Immunotools (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Il 10, by Immunotools (3 mentions)
Inf γ, by Immunotools (3 mentions)
Cd14 microbead, by Miltenyi Biotec (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Thermo Fisher Scientific (3 mentions)
Il 12, by Immunotools (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
73 protocols using interleukin 4 (il 4)
1
B Cell Isolation and Stimulation
2
Monocyte-Derived Dendritic Cell Activation by HSPs
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Monocytes were isolated by human monocyte enrichment cocktail (Stemcell Technologies, France). Isolated monocytes were cultured with recombinant human granulocyte macrophage colony‐stimulating factor and interleukin‐4 (IL‐4) (Immunotools, Germany), 50 ng/mL of each in RPMI complete media. At day 3, half of the media was replaced with fresh RPMI complete media with granulocyte macrophage colony‐stimulating factor and IL‐4. At day 6, 2×106/mL cells were cultured with or without HSP60 (MyBiosource) or HSP90 (HSPß90, Sigma Aldrich, MO) at the concentrations described. Cells were collected after 24 hours and analyzed with >90% cell viability. DC stained with CD11C‐PE, CD86‐Percp/Cy5.5, CD83‐APC, CD40‐fluorescein isothiocyanate (BD Bioscience), and HLA‐II‐fluorescein isothiocyanate (Biolegend) antibodies.
The endotoxin concentration in HSP60 was checked by LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific, IL). The level of endotoxin in the HSP60 was ≤1 EU/mg, according to manufacturer information, which is equal to ≤0.1 ng/mg. Our working concentration 5 μg/mL of HSP60 contains ≤0.0005 ng endotoxin. A similar concentration of endotoxin was determined in HSP90.
The endotoxin concentration in HSP60 was checked by LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific, IL). The level of endotoxin in the HSP60 was ≤1 EU/mg, according to manufacturer information, which is equal to ≤0.1 ng/mg. Our working concentration 5 μg/mL of HSP60 contains ≤0.0005 ng endotoxin. A similar concentration of endotoxin was determined in HSP90.
3
Monocyte-derived Dendritic Cell Culture
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Human primary cells were cultured at 37 C and 5% CO 2 in RPMI 1640 supplemented with gentamycin and 10% (v/v) heatinactivated fetal calf serum (complete medium). Human monocytes were purified from buffy coats by successive Ficoll and Percoll gradients. Monocyte-derived DCs were differentiated from monocytes by 5 days of culture with GM-CSF (50 ng ml À1 ; ImmunoTools) and IL-4 (10 ng ml À1 ; ImmunoTools), as described. 19 Proper differentiation was characterized by low CD14 and high CD1a and DC-SIGN expression levels.
4
Microglia Polarization Protocol
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Microglia were obtained from cerebral cortices of postnatal day 0–2 mice40 and treated for 24–48 h with LPS 100 ng/ml+IFNγ 20 ng/ml (ImmunoTools, Friesoythe, Germany) or IL-4 20 ng/ml (ImmunoTools) for cell polarization.
5
Differentiation of Human Monocytes
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Human CD14+ primary Mo were isolated from freshly prepared buffy coat (Karolinska Institutet Biobank, Stockholm, Sweden) using a pluriBead cell separation kit (pluriSelect GmbH, Leipzig, Germany). Mo were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) in six-well culture plates at a density of 1 × 106/ml and exposed to NhhA, as indicated, to induce differentiation. Control cells were primed with M-CSF (50 ng/ml) for 5 days. A subset of these cells was then polarized for 24 h by either IFN-γ (10 ng/ml) or LPS (20 ng/ml; Sigma-Aldrich, St. Louis, MO) to M1Mφ or IL-4 (20 ng/ml) to M2Mφ. To generate DCs, Mo were cultured in GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) for 5 days followed by LPS (20 ng/ml) for 2 days to enhance maturation. M-CSF and GM-CSF were purchased from ProSpec-Tany TechnoGene Ltd. (Israel). IL-4, IFN-γ, and IL-13 were purchased from ImmunoTools (Germany).
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
6
Generating Monocyte-Derived Dendritic Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat from healthy donors after informed consent as described previously [27 (link)]. Blood was drawn according to the instruction of our local ethics committee. To receive monocyte-derived DC, cells were separated through two density gradients using ficoll (Lymphoprep-Nycomed, Norway) and OptiPrep Density Gradient Medium (Sigma-Aldrich, München, Germany). Monocyte-derived DC were cultured in RPMI1640 medium supplemented with 10% heat-inactivated, autologous serum and 750 U/ml GM-CSF and 500 U/ml IL-4 (Immunotools, Friesoythe, Germany). The medium was replaced on day + 4 after the DC generation.
7
Monocyte-derived dendritic cell protocol
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Human monocytes were purified from buffy coats of healthy blood donors (EFS, Strasbourg, France) by successive Ficoll and Percoll gradients (Sigma-Aldrich). MonoDCs were differentiated from monocytes in 5 days with 200ng/mL GM-CSF (Schering-Plough) and 10ng/mL IL-4 (ImmunoTools), and characterized as CD14 low CD1a high DC-SIGN high .
8
Detailed Immunophenotyping of T-Cell Responses
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Goat anti-human Ig-PE (Southern Biotech), anti-PD-L1-APC (clone 29E.2A3, BioLegend), anti-EGFR-FITC (clone 528, Santa Cruz Biotechnology), anti-CD107a-APC (clone H4A3, BD Pharmingen), anti-CD137-PE (clone 4B4), anti-IFN-γ-PerCP-Cyanine5.5 (clone 4S.B3,), anti-CD3-PerCP-Cyanine5.5 (clone OKT-3, all from eBioscience), and anti-CD3-FITC (clone Ucht1), anti-CD8-FITC, APC (clone HIT8a), anti-CD56-PE (clone B-A19), anti-CD14-FITC, PE (clone MEM-15), anti-CD25-FITC, APC (clone MEM-181), anti-HLA-DR-FITC, PE (clone MEM-12), mouse IgG1-FITC, PE, Mouse IgG2b-APC, Annexin-V-FITC (all from Immunotools). Recombinant human IFN-γ, TNF-α, PGE2, GM-CSF, IL-1β, IL-4, IL-6, IL12 and anti-CD3 mAb UCHT-1 were from Immunotools. PD-L1-blocking mAb was from BPS Bioscience. Anti-EGFR mAb 425 was from Merck. Cetuximab was obtained from the Department of Hospital Pharmacy, UMCG, The Netherlands. Secretion of cytokines by T cells was measured using appropriate ELISA kits (IFN-γ from eBioscience and granzyme B from Mabtech).
9
Oleamide Modulation of Macrophage Polarization
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MDMs were cultured in RPMI 1640 supplemented with 10% human serum from buffy coats of healthy donors and 1% antibiotic-antimycotic (Gibco™, USA #15240062) at 37°C with 5% CO2. The M0 macrophages condition, monocytes were grown in a complete medium without stimulation for 6 days. In the M1 macrophages, monocytes were cultured in the presence of 50 ng/ml of GM-CSF (ImmunoTools GmbH, Germany) for 6 days. In the M2 macrophages, monocytes were cultured in 50 ng/ml M-CSF (ImmunoTools GmbH, Germany) for 6 days. Cell culture mediums were replaced every 3 days in all conditions. On day 6, M0 macrophages were incubated in complete medium alone for 24 h to serve as naïve macrophage or negative control cells. M1 macrophages were polarized with 20 ng/ml IFN-γ (ImmunoTools GmbH, Germany) and 10 ng/ml LPS of Escherichia coli O55:B5 (Sigma Aldrich, MO, USA) for 24 h. M2 macrophages were polarized using 20 ng/ml IL-4 (ImmunoTools GmbH, Germany) for 24 h. For experimental groups, M0, M1, and M2 macrophages (day 6) were cultured in desired conditions plus oleamide for 24 h.
10
Generating a CyTOF Reference Sample
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We generated a reference sample to use in each CyTOF run to adjust for batch effects, including differences in staining intensity between batches due to technical variations in the protocol or daily changes in instrument functioning. This reference sample contained PBMCs from 2 healthy controls of which half of the PBMCs were divided into equal amounts and stimulated with cytokines (IL-1b (10 ng/mL, PeproTech), IL-4 (10 ng/mL, ImmunoTools), LPS (10 ng/mL, Sigma), TGFb1 (100 ng/mL, Miltenyi), Dexamethasone (100 nM, Sigma), TNF (10ng/mL, PeproTech), IFN (10 ng/mL, PeproTech), PolyIC (500 ng/mL, Amersham Pharmacia Biotech, NJ, USA) and a mix of PMA (25 ng/mL, Sigma) and ionomycin (100 ng/mL, Sigma)) at 37 °C for 12 h to induce expression of all markers that were included in the CyTOF panel. For one of the donors, we isolated B cells following the manufacturer’s guidelines (EasySep Human B cell enrichment kit, Stem cell) to spike the reference sample to include more B cells. Unstimulated PBMCs, cytokine-stimulated PBMCs and isolated B cells were combined and stored in aliquots at –70 °C until further use.
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