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50 protocols using human il 4

1

Macrophage Polarization Pathway Analysis

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Phorbol-12-myristate-13-acetate (PMA) was bought from Sigma Aldrich (St. Louis, Missouri, USA), human IL-4 and human IL-13 were purchased from Peprotech (200-04 and 200-13, Rocky Hill, USA).The anti-CD163, anti-CD206, anti-β2-AR and anti-PKA antibodies were purchased from Santa Cruz Biotechnology(sc-33559, sc-58986, sc-271322 and sc-98951, Ca, USA). Phenylmethylsulfonyl fluoride (PMSF) was bought from beyotime biotechnology (Shanghai, China). Primary antibodies against STAT3, pSTAT3, pCREB were purchased from Cell Signaling Technology (4904s, 9131s and 9198s, Danvers, MA, USA). Anti-CREB antibody was purchased from Abcam (ab32515, Cambridge, MA, USA). AlexaFluor488 donkey anti-mouse antibody and AlexaFluor594 goat anti-rabbit antibody were purchased from ThermoFisher Scientific (A11037, A21202, Waltham, MA, USA). Fluorescein isothiocyanate dextran (FITC-dextran) was from Sigma Aldrich (FD40s, St. Louis, Missouri, USA). AlexFluor 647 was from Fcmacs (FMS-Msaf64701, Nanjing, Jiangsu, China).
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2

M2 Macrophage Differentiation Protocol

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Primary monocyte-derived macrophages were cultured with human IL-4 (20 ng/ml) on day 8 for 24 h for M2 differentiation, then total RNA was harvested. THP-1 cells were cultured in 1640 RPMI supplemented with 10% FBS and phorbol myristate acetate (PMA, 50 ng/ml, Sigma Aldrich, USA) for 24 h, then human IL-4 (20 ng/ml, PeproTech, USA) were added for 24 h to induce M2 differentiation before harvesting RNA. MH-S was exposed to mouse IL-4 (20 ng/ml, PeproTech, USA) for 48 h to induce M2 polarization.
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3

Differentiation of GFP⁺CD34⁺ HSCs into DCs

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Sorted GFP+CD34+ HSCs were allowed to expand for a further 72 h in X‐VIVO‐15/FCS/cytokine media at 2.5 × 104 mL−1 before differentiation into DCs according to a modified published protocol.30 In brief, the expanded cells were cultured at a density of 6.25 × 104 mL−1 in RPMI 1640, 10% FCS, 1 mm HEPES, 1 mm sodium pyruvate, 100 U mL−1 penicillin‐streptomycin, 2 mm Glutamax (all Thermo Fisher Scientific) and 50 µm β‐mercaptoethanol (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 100 ng mL−1 human FLT3L, 20 ng mL−1 human SCF, 2.5 ng mL−1 human IL‐4, 2.5 ng mL−1 human GM‐CSF (all cytokines from Peprotech) and 1 µm StemRegenin 1 (STEMCELL Technologies, Vancouver). Media and cytokines were replenished on Day 6 of the differentiation culture and harvested for analysis by flow cytometry after 12 days.
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4

Monocyte-Derived Dendritic Cell Activation

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Peripheral blood mononuclear cells (PBMC) from healthy donors (Sanquin) were isolated by centrifugation over a Ficoll gradient. Using magnetic CD14 Microbeads (Miltenyi), the CD14+ fraction was isolated from these PBMC. The CD14+ cells were cultured for 5 days in the presence of 800 U/ml human GM-CSF (Peprotech) and 500 U/ml human IL-4 (Peprotech). On day 5, the monocyte-derived DC (moDC; CD11c+ CD1a+ CD14 HLA-DRlo) were stimulated with the indicated reagents for 48 hours. The concentration of IL-12p40 in the supernatant after 24 hours was measured by ELISA (BioLegend). After 48 hours, the moDC were stained with fluorescently labeled antibodies targeted to CD83, CD86 and HLA-DR (eBioscience), and analyzed by flow cytometry (BD).
WT and TLR2-expressing HEK293 cells (Invivogen) were cultured in IMDM supplemented with 100 IU/ml penicillin/streptomycin (Gibco), 2 mM L-glutamin (Gibco) and 8% fetal calf serum (FCS; PAA Laboratories). TLR2-HEK293 cells were cultured in the presence of 10 μg/ml of the selective antibiotic blasticidin. In a 96 wells plate, 20,000 HEK293 or TLR2-HEK293 cells were seeded per well and stimulated with titrating doses of the indicated compounds in duplicates. After 24 hours of incubation at 37°C, supernatant was taken from all wells and the concentration of IL-8 was measured by ELISA (BioLegend).
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5

Cell Culture Reagents for Immunology Research

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Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 ATCC formulation, Hanks’ Balanced Salt Solution (HBSS) with calcium and magnesium, Dulbecco’s PBS, Penicillin/Streptomycin (10,000 U/mL), 1 M HEPES, 100× minimum essential medium (MEM) non-essential amino acids solution, 100× GlutaMAX supplement, 100 mM sodium pyruvate, Geneticin (50 mg/mL), BSA Fraction V 7.5% v/v solution, TrypLE, fibronectin, and versene were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Atlanta Biological (Flowery Branch, GA). Calcium 6 dye was purchased from Molecular Devices (San Jose, CA, USA).
For the CHO and dendritic cell (DC) experiments, X-vivo 15 medium was from Lonza (Basel, Switzerland). CaCl2, MgCl2, glucose, HEPES, 2% v/v human Ab serum, 7.5% w/v Na2HCO3, MEM (10×), FBS, Penicillin/Streptomycin, glutamine, prostaglandin E2 (PGE2), forskolin, formaldehyde, and fluoromount were from Sigma (St. Louis, MO, USA). Human IL-4, GM-CSF, TNFα, IL-1β, and IL-6 were from Peprotech (Rocky Hill, NJ, USA). Lymphoprep was from STEMCELL technologies (Vancouver, Canada). The PureCol bovine collagen I suspension was from Advanced Biomatrix (Carlsbad, CA, USA).
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6

IgE-mediated cell stimulation assay

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To use Ramos cells for stimulation, FcεRII expression was upregulated by stimulating with 20 ng/ml human IL4 (Peprotech) for 72 h. To bind IgE to Ramos cells and LAD2 cells, the cells were incubated with IgE for 1 h on ice, and IgE binding was confirmed with flow cytometry using IgE-specific antibodies. For Jurkat cell stimulation, 0.125 × 106 Jurkat cells were mixed with equal numbers of U266 cells, Ramos cells with IgE bound, or LAD2 cells with IgE bound in 200 μl of medium, incubated for 5 h at 37°C, and stained with anti-CD69 antibodies for flow cytometry analysis. To distinguish CAR+ Jurkat cells from stimulator cells, in addition to anti-CD69-APC, anti-FcεRI-PE was used for co-culture with U266 or Ramos cells to label CAR+ Jurkat cells. Since LAD2 cells express FcεRI, to separate LAD2 cells and CAR+ Jurkat cells in co-cultures, anti-CD117-PE was used to label LAD2 cells. To distinguish Daudi or Daudi-IgE cells with Jurkat cells in co-cultures, Jurkat cells were first labeled with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) by incubating with 0.3 μM CFSE in DPBS-5% FBS for 5 min at room temperature (27 (link)).
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7

Immunoblotting Antibody Panel Validation

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The following antibodies were used for immunoblotting. Abcam: anti-PMP70 (1:1,000 dilution; ab3421), anti-FASN (1μg/ml dilution; ab22759); Cell Signaling: anti-cytochrome oxidase IV (1:250 dilution; 4850), anti-catalase (1:800 dilution; 12980), anti-centromere protein A (1:400 dilution; 2186), anti-calreticulin (1:200 dilution; 12238), anti-FASN (1:1,000 dilution; 3180); Santa Cruz Biotechnology: anti-C13orf31 (1:200 dilution; sc-374553; E7 and 1:500 dilution; sc-376231; E12), anti-caspase 1 p20 (1:250 dilution; sc-1218-R); R&D Systems: anti-IL-1β (1:250 dilution; AF-401-NA); Sigma: anti-β-actin (1:10,000 dilution; A5060). All antibodies used have validation profiles on either Antibodypedia or 1DegreeBio.
The following reagents were used: M-CSF (Peprotech, 300-25), LPS (from Escherichia coli K12, InvivoGen, tlrl-peklps), human IFN-γ (Peprotech, 300-02), murine IFN-γ (Peprotech, 315-05), human IL-4 (Peprotech, 200-04), murine IL-4 (Peprotech, 214-14), ATP (Sigma Aldrich, A2383), C75 (Sigma Aldrich, C5490), etomoxir (Sigma Aldrich, E1905), MitoTEMPO (Sigma Aldrich, SML0737), oligomycin (Sigma Aldrich, O4876), FCCP (Sigma Aldrich, C2920), rotenone (Sigma Aldrich, R8875), antimycin A (Sigma Aldrich, A8674), palmitate-BSA (Seahorse Bioscience, 102720-100), zymosan A (Sigma Aldrich, Z4250), PMA (Sigma Aldrich, P1585), HRP (Sigma Aldrich, P8375), luminol (Sigma Aldrich, A8511).
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8

Lipid Extraction and Analysis Protocol

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PMA,LPS, and formic acid were purchased from Sigma-Aldrich. Mouse IL-4, human IL-4, and human IL-13 were obtained from PeproTech. High-performance liquid chromatography (HPLC)-grade chloroform, methanol, isopropanol (IPA), and hexane were used without further purification. Phosphoricacid, potassium chloride, and ammonium acetate were obtained from Sinopharm Chemical Reagent Co., Ltd. The water was purified using a 0.22-µm Milli-Qfilter (Millipore, USA).
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9

Isolation and Culture of Monocytes

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Blood was collected during exsanguination. Some of the peripheral blood leukocytes were subjected to flow cytometry in order to evaluate the frequency of CD3, CD19, CD33, CD45, and BDCA-3 positive cells. Remaining blood, spleen, and bone marrow were used for an independent separation of intact monocytes using the negative section with the use of magnetic beads as described before (17 (link), 27 (link)). The purity of the cells exceeded 95% for CD33(bright) and 80% for CD14(+). Isolated MO were incubated in X-VIVO 10/15™ media with gentamycin and phenol red (Lonza, Cohasset, MN, USA), supplemented with human IL-4 (Peprotech, Rocky Hill, NJ, USA) at 500 IU/ml and human GM-CSF (Peprotech, Rocky Hill, NJ, USA) at 1,000 IU/ml at 37°C and 5% CO2. On day 3, 50% of the supernatant was collected and replenished with fresh media and cytokines. After 5 days, the supernatants and cells were collected using an ice-cold 10 mM EDTA buffer (Gibco, Grand Island, NY, USA), followed by a washout with PBS without Ca2+ or Mg2+ (Gibco, Grand Island, NY, USA) (27 (link)). In some experiments, rabbit polyclonal neutralizing antibody for M-CSF (FN M-CSF, 5 µg/ml) was added at the beginning of the culture. The dose was shown to fully neutralized M-CSF in prior study (17 (link)). An addition of non-specific IgG had no effect on MO function (17 (link)).
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10

Macrophage Polarization and Treatment

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Isolation, polarization and treatment of macrophages with a CD64-specific IT or a corresponding human cytolytic fusion protein (hCFP) was carried out as previously described [33 (link), 34 (link)]. Briefly, PBMCs were isolated from buffy coats (Western Province Blood Transfusion Service, Cape Town, South Africa) and cultured for 3 hours in serum free media to select for monocytes by adherence. Monocytes were afterwards seeded at 1 x 105 cells/well in 12 well plates and polarized for 72 h using 100 U/ml human IFN-γ (Sigma-Aldrich, St. Louis, MO, USA) and 1 μg/ml LPS (Sigma-Aldrich) for M1 and 20 ng/ml human IL-4 (Peprotech, Hamburg, Germany) for M2 macrophages. The polarization was boosted after 72 h with half of the initial stimulus for 24 h. Successful polarization of macrophages was confirmed by microscopic observation of cell morphology and fluorescent analysis of cell surface receptors. Thereafter, polarized macrophages were incubated with 200 nM hCFP and 100 nM IT. Negative control cells were treated with PBS. After 24 h, apoptosis assay was carried out as described above except that PI was replaced with 7-amino-actinomycin D (7-AAD) as a marker of viable cells. Before commencement of study, the protocol was approved by the Human ethics research committee of the University of Cape Town, South Africa.
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