Single‐cell splenocyte suspensions were obtained by passing spleen samples through a 70‐µm nylon cell strainer using a syringe. The splenocyte suspension was rinsed with RPMI 1640 medium (Lonza) and incubated with lysis buffer (8.3 g NH4Cl, 1 g KHC3O and 37.2 mg EDTA dissolved in 1 L demi water, filter sterilized) to remove red blood cells. The reaction was stopped by adding RPMI 1640 medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Bodinco), penicillin (100 U/mL)/streptomycin (100 µg/mL; Sigma‐Aldrich) and β‐mercaptoethanol (20 µmol/L; Thermo Fisher Scientific). Splenocytes were subsequently resuspended in this culture medium. For the ex vivo antigen‐specific restimulation assay, splenocytes (8 × 105 cells/well) were cultured in culture medium with or without 500 µg/mL CMP/hWP/nWP. Supernatant was harvested after 4 days of culture (37°C, 5% CO2) and stored at −20°C until cytokine analysis. Measurements of IL‐5, IL‐13, IL‐10 and IFNγ were performed by means of ELISA according to the protocol described above for IgE. Purified rat anti‐mouse antibodies (1 µg/mL for IL‐5 and IFNγ and 2 µg/mL for IL‐13 and IL‐10), recombinant mouse cytokines and biotinylated rat anti‐mouse antibodies (1 µg/mL for IL‐5, IL‐10 and IFNγ and 400 ng/mL for IL‐13), were purchased at BD Biosciences.
Recombinant mouse cytokines
Manufactured by BD
Recombinant mouse cytokines are proteins produced through recombinant DNA technology that are derived from mouse cells. They are used in research applications to study the biological functions and signaling pathways of cytokines in the mouse model.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated rat anti mouse antibodies, by BD (2 mentions)
Ifn γ, by BD (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse antibodies, by BD (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Interleukin 4 (il 4), by BD (1 mentions)
Microplate autoreader, by Agilent Technologies (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Il 13, by BD (1 mentions)
Il 17a, by Thermo Fisher Scientific (1 mentions)
Tnf α, by BioLegend (1 mentions)
3 protocols using recombinant mouse cytokines
1
Isolation and Cytokine Analysis of Murine Splenocytes
2
Cytokine Quantification by ELISA
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The cytokine levels in culture supernatants were measured by sandwich ELISA methods. Briefly, the anticytokine antibody, including purified rat anti-mouse cytokine monoclonal antibodies IL-2, IFNγ, IL-4, and IL-5 (PharMingen, San Diego, CA), were coated in the 96-well plates (Nunc, Roskilde, Denmark). After overnight incubation at 4°C and blocking with PBS containing 1% BSA for 30 min, the samples and standards (recombinant mouse cytokines, PharMingen) were added to the 96-well plates for 2 hours of incubation. The biotin-conjugated antibodies (biotinylated rat anti-mouse cytokine monoclonal antibodies, PharMingen) were added and incubated. After washing, the streptavidin-conjugated peroxidase (Thermo Fisher Scientific Inc., Rockford, IL) was added for 1 hour. The substrates, 2,2′-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid (ABTS, Sigma), were added to each well for 20 min to develop color. The plates were read in a Microplate autoreader (Microplate autoreader; Bio-Tek Instrument, Inc. Winooski, VT) at 405 nm. The detection limits for IL-2, IFNγ, and IL-5 are 75 pg/mL and 3.9 pg/mL for IL-4.
3
Splenocyte-BMDC Co-culture Cytokine Assay
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Spleens were aseptically removed and single cell suspensions were obtained as described in Section 2.2.4 . Splenocytes (5 × 106) were co-cultured with BMDC (5 × 105) either or not loaded with Influvac in U-bottom 96-well plates at 37 °C, 5% CO2. After 5 days of incubation, supernatants were collected and stored at −20 °C for cytokine analysis. IFNγ, IL-13 (both from R&D) were measured by means of ELISA according to the protocol described in Section 2.1.4 . for galectin-4 and -9. Purified rat anti-mouse antibodies (1 µg/mL for IFNγ and 2 µg/mL for IL-13), recombinant mouse cytokines and biotinylated rat anti-mouse antibodies (1 µg/mL for IFNγ and 400 ng/mL for IL-13) were purchased from BD Biosciences. TNFα (Biolegend), IL-10 and IL-17A concentrations (both from Thermo fisher) were measured by ELISA according to manufacturer’s protocol.
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