Interleukin (IL)-1α, IL-1β, and interferon- beta (IFN-β) protein production was determined using commercially available AlphaLISA kits (Perkin Elmer, Waltham, MA, USA) in cell-free culture supernatant. Similarly, IL-8 (BD Biosciences, San Diego, CA, USA), IL-1 receptor antagonist (IL-1Ra), soluble IL-1 receptor 2 (sIL-1R2), C-X-C motif chemokine 10 (CXCL10), Chemokine (C-C motif) ligand 5 (CCL5), IL-28A, IL-28B, and IL-29 protein production (R&D Systems, Minneapolis, MN, USA) were all determined using commercially available ELISA kits performed according to manufacturer's instructions. Samples below the detection range were arbitrarily reported as half the lower limit and included in the analysis with all other samples as previously described (21 (link)).
Il 28b
Manufactured by R&D Systems
Sourced in United States
IL-28B is a recombinant human protein that functions as a cytokine. It is a member of the type III interferon family and is involved in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Il 28a, by R&D Systems (2 mentions)
Il 29, by R&D Systems (2 mentions)
Alphalisa kit, by PerkinElmer (1 mentions)
Cxcl10, by R&D Systems (1 mentions)
Panserin 401, by PAN Biotech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Il 27, by R&D Systems (1 mentions)
Pma ionomycin, by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by GE Healthcare (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Hil 6, by Merck Group (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Gentamycin, by Roche (1 mentions)
Ifn γ imukin, by Boehringer Ingelheim (1 mentions)
Ifn α2b intron a, by Merck & Co (1 mentions)
Hil 2, by Roche (1 mentions)
Il 21, by R&D Systems (1 mentions)
5 protocols using il 28b
1
Cytokine Protein Quantification Assay
2
Cell Culture Conditions for Diverse Immune Cells
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EBV–B cell lines were cultured in RPMI (Invitrogen), and SV40-fibroblasts and 293T HEK cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Saimiri T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN-Biotech GmbH), supplemented with 10% FBS, 350 µg/ml glutamine, 100 µg/ml gentamycin, and 10 U/ml hIL-2 (Roche). PBMCs were isolated from blood by Ficoll-Hypaque density gradient centrifugation (GE Healthcare) and cultured at a density of 106 cells/ml in RPMI supplemented with 10% FBS. Stimulations were performed, at the doses stated and for the indicated times, with IL-12 (R&D Systems), BCG (donated by C. Nathan, Weill Cornell Medical College, New York, NY), IL-18 (R&D Systems), PMA/ionomycin (Sigma-Aldrich), IL-2 (BD), IL-23 (R&D Systems), IFN-γ (Imukin; Boehringer Ingelheim), IL-27 (R&D Systems), IFNα-2b (Intron A; Schering Plough), IFNβ-1b (PeproTech), LPS (Sigma-Aldrich), IL-21 (R&D Systems), IL-29 (R&D Systems), IL-28B (R&D Systems), IL-10 (R&D Systems), IL-6 (R&D Systems), hyper–IL-6 (hIL-6; donated by S. Rose-John), and LIF (EMD Millipore). hIL-6 was prepared as described elsewhere (Fischer et al., 1997 (link)).
3
Regulatory T-cell Induction Assay
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Spleens were removed aseptically from the euthanized normal female BALB/c mice. The splenocytes were prepared with the lymphocyte separation medium as described above. Collected cells were washed twice with RPMI 1640 and resuspended in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, MO, USA) for culturing experiments. Cell viability was evaluated by the trypan blue dye exclusion test.
Splenocytes (5 × 106 cells/ml) in quintuplicate were seeded in 96-well plates supplemented with RPMI 1640 complete medium in a final volume of 200 μl. The cells were cultured with anti-CD3/anti-CD28 antibody (Abs) (both from eBioscience, San Diego, USA), IL-2 (PeproTech, London, USA), and transforming growth factor-β (TGF-β) (R&D Systems, MN, USA) in the presence or absence of IL-28B (5, 50, and 400 ng/ml) (R&D Systems, MN, USA) were added to the culture medium. Three days later, cells were harvested and analyzed for CD4, CD25, and Foxp3 expression by flow cytometry.
Splenocytes (5 × 106 cells/ml) in quintuplicate were seeded in 96-well plates supplemented with RPMI 1640 complete medium in a final volume of 200 μl. The cells were cultured with anti-CD3/anti-CD28 antibody (Abs) (both from eBioscience, San Diego, USA), IL-2 (PeproTech, London, USA), and transforming growth factor-β (TGF-β) (R&D Systems, MN, USA) in the presence or absence of IL-28B (5, 50, and 400 ng/ml) (R&D Systems, MN, USA) were added to the culture medium. Three days later, cells were harvested and analyzed for CD4, CD25, and Foxp3 expression by flow cytometry.
4
Modulating Antiviral Immunity Against EV-A71
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Neutralizing antibodies against mouse IFN- alpha receptor or IFN- gamma (Bio X Cell) were i.p. injected at a dose of 150 μg/mouse into 1-week-old wt-129 mice, either before or after i.p. inoculation with EV-A71 clinical isolate F23 (108 PFU/mouse). The neutralizing antibody against the IFN-alpha receptor was injected 1 day before EV-A71 inoculation, followed by repetitive antibody treatments on 1 and 3 dpi. Recombinant mouse IL28A and IL28B were both purchased from R & D Systems®. Various doses of mouse IL28 was i.p. injected into mice at dpi = 1 (Additional file 4 : Figure S4a). Recombinant mouse IFNαA (PBL Assay Science Co.), GS9620 (Cayman Chemical) and R837 (InvivoGen), were orally or i.p. injected into wt-129 mice infected with EV-A71, at the dose of 300 IU/g (mouse IFNαA), 3.0 μg/g (GS-9620), 4.5μg/g (GS-9620), and 5.0 μg/g (R837), respectively.
5
Differentiation of Airway Epithelial Cells
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Soluble interferons were used at 30 ng/mL for IL-28A (R&D Systems), IL-28B (R&D Systems) and IL-29 (Gibco). For IFN-β (pbl Interferon Source) 2.5 ng/mL was used. Mediator was present in both apical and basal media for week one, and in the basal media once brought to ALI at day 6. Media was replaced three times a week and fresh mediator added. The experiment was terminated after 21 days by fixation using in 4% paraformaldehyde. Fixed cells were stained for epithelial composition using antibodies specific to acetylated α-tubulin, MUC5AC and MUC5B.
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