Ifn α2

Manufactured by BioLegend

Sourced in United States

IFN-α2 is a recombinant human interferon alpha-2 protein. Interferons are a class of signaling proteins involved in the body's immune response to pathogens.

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Lab products found in correlation

Tnf α, by BioLegend (2 mentions) Cytofix fixation buffer, by BD (1 mentions) Legendplex assay, by BioLegend (1 mentions) Legendplex human anti virus response panel, by BioLegend (1 mentions) Elisa, by R&D Systems (1 mentions) Osteopontin, by Bio-Rad (1 mentions) 3p hprna, by InvivoGen (1 mentions) Tlrl hprna, by InvivoGen (1 mentions) Lyec 1, by InvivoGen (1 mentions) Plad b, by Bio-Techne (1 mentions) Canto 1, by BD (1 mentions) Il8 cxcl8, by BioLegend (1 mentions) Mcp 1 ccl 2, by BioLegend (1 mentions) Interleukin 6 (il 6), by BioLegend (1 mentions) Ifn γ, by BioLegend (1 mentions) Il 1β, by BioLegend (1 mentions) Ifn β, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Il 21, by Thermo Fisher Scientific (1 mentions)

6 protocols using ifn α2

PBMC Stimulation and STAT Phosphorylation

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PBMCs were thawed and cultured overnight as described above. The following day, 1.0 × 10⁶ viable PBMCs were stimulated with or without 300 ng/ml of IFN-α2 (BioLegend, San Diego, CA, USA) in RPMI complete medium for 20 min at 37°C and agitation at 300 rpm. During the stimulation period, cells were stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, anti-human lineage cocktail 3, and anti-CD56-V450. Thereafter, cells were fixed with the BD Cytofix Fixation Buffer for 30 min at 4°C. Then, cells were washed twice and resuspended in PBS supplemented with 2% FBS. Finally, cells were stained with anti-STAT1-PE and anti-pSTAT1 (pY701)-PerCPCy5.5 or anti-pSTAT4(pY693) during 60 min at room temperature.

Perpiñán E., Pérez-Del-Pulgar S., Londoño M.C., Mariño Z., Bartres C., González P., García-López M., Pose E., Lens S., Maini M.K., Forns X, & Koutsoudakis G. (2020). Cirrhosis Hampers Early and Rapid Normalization of Natural Killer Cell Phenotype and Function in Hepatitis C Patients Undergoing Interferon-Free Therapy. Frontiers in Immunology, 11, 129.

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Comprehensive Cytokine Profiling of Cell Culture

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In total, 43 human soluble factors were measured in the culture media harvested from three independent proliferation assays using a 37-parameter Bio-Plex™ Human Inflammation Panel including IL-27(p28), gp130/sIL-6Rβ, IL-34, IL-22, sIL-6Rα, IFN-α2, IFNγ, IL-26, MMP-2, IL-12(p40), IL-19, IL-20, IL-29(IFN-λ1), IL-35, IL-32, BAFF/TNFSF13B, IL-2, IL-11, APRIL/TNFSF13B, MMP-1, IFN-β, MMP-3, sCD163, Pentraxin-3, LIGHT/TNFSF14, TSLP, sCD30/TNFSF8, IL-8, IL-10, TWEAK/TNFSF12, Osteocalcin, IL-28A/(IFN-λ2), sTNF-R2, chitinase-3-like1, sTNF-R1, IL12(p70), and Osteopontin (Bio-Rad) and a 13-parameter LEGENDplex™ Human Anti-Virus Response Panel consisting of IL-1β, IL-6, TNF-α, IP-10 (CXCL10), IFN-λ1(IL-29), IL-8, IL12(p70), IFN-α2, IFN-λ2/3(IL28-A/B), GM-CSF, IFN-β, IL-10, and IFNγ (Biolegend), according to manufacturers' protocol. The overlapping factors between the two kits were adjusted. Levels of human soluble M-CSF were determined by ELISA (R&D systems). Concentrations of CXCL9 and CXCL10 in sera of treated or control mice were analyzed by a Legendplex assay (Biolegend).

Eissler N., Mao Y., Brodin D., Reuterswärd P., Andersson Svahn H., Johnsen J.I., Kiessling R, & Kogner P. (2016). Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade. Oncoimmunology, 5(12), e1232222.

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Cell-Based Cytokine Induction Assay

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Plad B was purchased from Tocris (6070), and spliceostatin analog PF-06437837 (thailanstatin A methyl ester) was synthesized by Pfizer as previously reported (22 (link)). Cells were seeded in 96-well plates at 5000 cells/well (4T1), 10,000 cells/well (A549, CT26, and MC38 HEK), and 20,000 cells/well (B16F10) overnight in 75 μl media. The next day, 75 μl of a 2× solution of the indicated compound was added to wells, and cells were incubated at 37 °C until the indicated time points. Assays with THP1 cells were performed similarly except plating cells (100,000 cells) immediately before the addition of indicated compounds. Supernatant was removed and used in subsequent assays. 3p-hpRNA (Invivogen; tlrl-hprna) was prepared as a 2× solution in complex with the transfection reagent LyoVec (Invivogen; lyec-1) according to the manufacturer's instructions. After complexing, 75 μl of the 2× concentrated 3p-hpRNA/LyoVec was added to cells until the indicated time points. Positive control cytokines used were IFN-α2 (Biolegend; 592702) and TNFα (Biolegend; 570102).

Chang A.Y., Zhou Y.J., Iyengar S., Pobiarzyn P.W., Tishchenko P., Shah K.M., Wheeler H., Wang Y.M., Loria P.M., Loganzo F, & Woo S.R. (2021). Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response. The Journal of Biological Chemistry, 297(5), 101277.

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Quantifying Cytokines and IgG Isotypes in NLF

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A 13-multiplex cytometric CBA (IL1β, IFNα2, IFNγ, TNFα, MCP1/CCL2, IL6, IL8/CXCL8, IL10, IL12p70, IL17A, IL18, IL23 and IL33: Biolegend, CA, USA), and human IgG isotype bead array (IgG1, IgG2, IgG3 and IgG4: Biolegend) were used to quantify the cytokines and IgG isotypes in NLF samples. The determinations were performed in duplicate according to the manufacturer´s instructions. Samples were run on a CANTO I (BD Biosciences, San José, CA, USA) cytometer and the calibration curves were above r² 0.97.

Cortegano I., Rodríguez M., Hernángómez S., Arrabal A., Garcia-Vao C., Rodríguez J., Fernández S., Díaz J., de la Rosa B., Solís B., Arribas C., Garrido F., Zaballos A., Roa S., López V., Gaspar M.L, & de Andrés B. (2022). Age-dependent nasal immune responses in non-hospitalized bronchiolitis children. Frontiers in Immunology, 13, 1011607.

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P2RY8 Expression in Stimulated PBMCs

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Thawed PBMCs were washed twice and resuspended in RPMI 1640 medium containing 10% FBS, 10 mM Hepes, 55 µM β-mercaptoethanol, 2 mM glutamine, and 50 IU penicillin/streptomycin. Cells were plated at 3×10⁵ cells/96-well and stimulated with IL-6 (20 ng/ml; Peprotech; Cat #200-06), IL-2 (100 U/ml; Peprotech; Cat #200-02), IL-21 (20 ng/ml; Peprotech; Cat #200–21), ETP (50 µM; MedChemExpress; Cat #HY13629), IFN-α2 (50 ng/ml; BioLegend; Cat #592702), IFN-β (50 ng/ml; Peprotech; Cat #200-06), IFN-γ (20 ng/ml; Peprotech; Cat #300-02), LPS (1 µg/ml; Sigma-Aldrich; Cat #L6529), αIgA, G, M (5 µg/ml, Jackson ImmunoResearch; Cat #109-006-064), GGG (100 nM), IL-10 (50 ng/ml; Peprotech; Cat #200-10), R837 (5 µg/ml; InvivoGen; Cat #tlrl-imqs), or CPG-A (0.5 µM; InvivoGen; Cat #tlrl-2216). P2RY8 expression was analyzed by flow cytometry after 24 h or 48 h of stimulation.

He Y., Gallman A.E., Xie C., Shen Q., Ma J., Wolfreys F.D., Sandy M., Arsov T., Wu X., Qin Y., Zhang P., Jiang S., Stanley M., Wu P., Tan J., Ding H., Xue H., Chen W., Xu J., Criswell L.A., Nititham J., Adamski M., Kitching A.R., Cook M.C., Cao L., Shen N., Cyster J.G, & Vinuesa C.G. (2021). P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance. The Journal of Experimental Medicine, 219(1), e20211004.

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ZIKV Infection Cytokine Response

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The HRvEC and HUVEC cells were pretreated with AICAR for 1h followed by ZIKV infection (MOI 1) for 48 h. The protein levels of IFNα2, TNFα and IL-6 were quantified in conditioned media using ELISA. ELISA was performed for IFNα2 (BioLegend, San Diego, CA, sensitivity 3 pg/mL) and inflammatory cytokines TNFα and IL-6 (R&D Systems, Minneapolis, MN) as per manufacturer’s instruction and the data were represented as pg/mL of culture media. The experiment was performed in biological triplicates and technical duplicates for statistical analysis.

Singh S., Singh P.K., Suhail H., Arumugaswami V., Pellett P.E., Giri S, & Kumar A. (2020). Adenosine monophosphate-activated protein kinase (AMPK) restricts Zika virus replication in endothelial cells by potentiating innate antiviral responses and inhibiting glycolysis. Journal of immunology (Baltimore, Md. : 1950), 204(7), 1810-1824.

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