Blood was collected from healthy cattle in 60 ml syringes containing 7.5% EDTA. PBMCs were obtained from these blood samples using the Ficoll (GE Healthcare Bioscience) method of isolation.21 CD14+ monocytes were isolated from PBMCs by positive selection using MACs columns (Miltenyi Biotec Inc.). Mature bDCs were subsequently derived from CD14+ monocytes after culturing in a complete Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% heat-inactivated FBS and 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (Hyclone Laboratories Inc.), 2-mercaptoethanol (Sigma-Aldrich), recombinant bovine GM-CSF at 100 ng/ml (Bio-Rad) and IL-4 at 50 ng/ml (Biorad) as previously described.22 (link) bDCs were assessed by flow cytometry and the expression of DC-specific marker CD206 was found to be greater than 97%.
Interleukin 4 (il 4)
Manufactured by Bio-Rad
Sourced in United States
IL-4 is a recombinant protein produced by Bio-Rad. It is a cytokine that plays a role in the regulation of immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Il 10, by Bio-Rad (5 mentions)
Ifn γ, by Bio-Rad (5 mentions)
Interleukin 6 (il 6), by Bio-Rad (5 mentions)
Interleukin 2 (il 2), by Bio-Rad (4 mentions)
Tnf α, by Bio-Rad (4 mentions)
Gm csf, by Bio-Rad (3 mentions)
Il 23, by Thermo Fisher Scientific (2 mentions)
Ifn λ1, by Thermo Fisher Scientific (2 mentions)
Ifn λ2, by Thermo Fisher Scientific (2 mentions)
Ifnλ3, by Thermo Fisher Scientific (2 mentions)
Ifnλ4, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Il 17a, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Bio-Rad (2 mentions)
Il 17a, by Bio-Rad (2 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
4 2 hydroxyethyl 1piperazineethanesulfonic acid hepes, by Cytiva (1 mentions)
Il 17a, by Merck Group (1 mentions)
7 protocols using interleukin 4 (il 4)
1
Isolation and Differentiation of Bovine Dendritic Cells
2
Interictal Plasma Cytokine Profiling
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Interictal samples were collected at day 7 from the last seizure attack. The plasma was harvested within 30 min at 37 °C of venipuncture from EDTA-anticoagulated blood samples and stored at −80 °C for subsequent cytokine analysis. The concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, IFNγ, GM-CSF, TNFα (Bio-Rad, USA), IL-17a (PeproTech, Rocky Hill, NJ, USA), IL-1β (Bender MedSystems, Vienna, Austria), IL1Ra (Cytoscreen, Biosource, Belgium), IFNλ1, IFNλ2, IFNλ3, IFNλ4 (eBioscience, CA, USA), and IL-23 (Invitrogen, Carlsbad, CA, USA) were measured by ELISA according to manufacturers’ instruction [16 (link)–20 (link)]. CSF concentrations of IL-6, IFNγ, IFNλ3 and IL-17a were measured using human cytoline/chemokine MILLIplex kits (Millipore Corp, Billerica, MA, USA) [21 (link)]. ELISAs were performed in duplicate.
3
Cytokine Profile Analysis in Plasma
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Blood samples were collected by venipuncture into EDTA-anticoagulation vials. Plasma was harvested from the collected samples within 30 min and stored at −80 °C for subsequent cytokine analysis. Concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, IFNγ, GM-CSF, TNFα (all antibodies from Bio-Rad, USA), IL-17a (PeproTech, Rocky Hill, NJ, USA), IL-1β (Bender MedSystems, Vienna, Austria), IL1Ra (Cytoscreen, Biosource, Belgium), IFNλ1, IFNλ2, IFNλ3, IFNλ4 (eBioscience, CA, USA), and IL-23 (Invitrogen, Carlsbad, CA, USA) were measured by ELISA according to manufacturers’ instruction16 (link)22 (link)23 (link). ELISAs were performed in duplicate.
4
Multiplex Cytokine Profiling in Serum
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Serum cytokine levels were analyzed using single-plex sets for IL-1b, IL-2, IL-5, IL-4, IL-6, IL-8, IL-10, IL-12p40, GM-CSF, IFN-γ, CXCL10, and TNF-α (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. Serum aliquots (50 μL) were used for analysis, with a minimum of 50 beads acquired per analyte. Median fluorescence intensities were measured using a Luminex 200 analyzer. Data collected was analyzed with MasterPlex CT control software and MasterPlex QT analysis software (Hitachi Software, San Bruno, CA, USA). Standard curves for each analyte were generated using standards provided by the manufacturer.
5
Multiplex Analysis of Immune Biomarkers
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Biomarkers associated with inflammation, macrophage, and lymphocyte activation were measured by magnetic bead-based multiplex according to the manufacturer’s instructions, including APRIL/TNFSF13, BAFF/TNFSF13B, sCD163, IL-2, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L, and TNF-α (Bio-Rad, Hercules, CA). Multiplex results were analyzed with Bio-Plex Manager Software (Bio-Rad, Hercules, CA). sCD14 was measured by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) (18 (link)) and analyzed using Tecan’s Magellan™ Software (Tecan US, Morrisvillle, NC).
6
Bronchoalveolar Lavage Cytokine Analysis
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Mice were euthanized using CO2 and their lungs were surgically exposed. The trachea was cannulated and the lungs were lavaged with 5 aliquots of 800 µL of 0.9% saline (VWR International, West Chester, PA). Bronchoalveolar lavage (BAL) fluid supernatant was stored at −80°C until further use. BAL fluid total protein was quantified with a total protein assay kit using Bicinchoninic Acid (Thermo Scientific, Waltham, MA) and Granzyme B was measured using a specific ELISA kit (R&D systems, Minneapolis, MN). Cytokine analysis was performed on the BAL fluid using mouse 23-plex and 9-plex cytokine assays that included detection of IFN-γ, IL-4, IL-5, IL-13, IL-17, IL-10, IL-15, and CCL5 (Bio-Rad Laboratories, Hercules, CA).
7
Cytokine Profiling of Stimulated Cells
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An additional 330 μL aliquot of the femur and soft tissue single cell suspensions was taken and cells stimulated with 50 ng mL -1 phorbol 12-myristate 13-acetate (PMA, Sigma, Switzerland) and 500 ng mL -1 ionomycin (Sigma, Switzerland). Following incubation for 24 h at 37 °C, 5% CO 2 , 50 μL of the cell culture supernatants were analyzed using a customized Bioplex kit for the following cytokines: IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17A, IFN-γ and keratinocyte chemoattractant (KC), (BIORAD, Switzerland). Analysis was performed using a BIO-PLEX 200 system connected to a BIO-PLEX with high-throughput fluidics (BIORAD, Switzerland).
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