Ifnα2a

Manufactured by Thermo Fisher Scientific

IFNα2a is a recombinant human interferon alpha 2a, a cytokine protein that is used in various research and clinical applications. It functions as an antiviral, antiproliferative, and immunomodulatory agent.

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5 protocols using ifnα2a

Analyzing Antiviral Signaling Pathways

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Recombinant human TNFα, IFNα2a, IFNβ, and
IFNλ1, and IFNλ2 were purchased from Peprotech and prepared
according to the manufacturer’s recommendations. Stocks were prepared as
single-use aliquots and stored at −80°C. Ruxolitinib was
purchased from Selleckchem and used at a final concentration of 3 μM.
Danoprevir, an inhibitor of HCV’s NS3-4A protease, was purchased from
Selleckchem and used at 2 μM. Etanercept (Amgen) was kindly provided by
the Kennedy Institute of Rheumatology and was stored in single-use aliquots at
4°C. Anti-CD81 (clone JS81, no azide-low endotoxin) was purchased from
BD Biosciences. The viral IFN antagonists B18R^{34 (link)} and 136R^{35 (link)} were purchased from Affymetrix and
Biotechne respectively. Cytokines, antagonists, and drugs were replaced at each
media change unless otherwise indicated.

Laidlaw S.M., Marukian S., Gilmore R.H., Cashman S.B., Nechyporuk-Zloy V., Rice C.M, & Dustin L.B. (2017). Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent from Interferons. Gastroenterology, 153(2), 566-578.e5.

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Propagation and Maintenance of Huh7 Cell Lines

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Huh7 and Huh7.5-SEC14L2 cells (gift from P. Simmonds, Oxford) were propagated in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL streptomycin, 100 U/mL penicillin, and 2 mmol/L L-glutamine (all from Thermo Fisher, Waltham, MA). Huh7.5-SEC14L2 cells expressing HCV G3 S52 WT and A150V replicon²⁵ (link) were maintained in the presence of 750 μg/mL G418 (Sigma-Aldrich, St Louis, MO). 2-AP is from Sigma-Aldrich. IFNα2a was from PeproTech (Rocky Hill, NJ), and sofosbuvir was provided by Gilead Sciences, Foster City, CA. Polyinosinic-polycytidylic acid (1:6 w/w, high molecular weight, 1.5–8 kb) conjugated with LyoVec and 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) are from InvivoGen (San Diego, CA).
Infectious G3 cell-culture-derived HCV was generated as detailed previously.¹⁶ (link)

Lee W.Y., Jones M., Wing P.A., Rajagopal S, & Foster G.R. (2020). The A150V Polymorphism of Genotype 3 Hepatitis C Virus Polymerase Inhibits Interferon Alfa by Suppressing Protein Kinase R Activation. Cellular and Molecular Gastroenterology and Hepatology, 11(4), 1163-1175.

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Quantifying IFN-induced gene expression

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For IFN stimulation, 7.5 × 10⁵ cells/12-well (RNA samples for quantitative real-time reverse transcription PCR (RT-qPCR)) or > 5 × 10⁵ cells/12-well (whole-cell lysates for immunoblotting) were seeded. MDMs were left untreated or treated with 1000 U/mL IFNα2a (PeproTech), IFNβ1a (PBL Interferon Source) or IFNγ (PeproTech) for 8/24 h (RNA samples) or 24 h (whole-cell lysates) at 37 °C. As a control for IFN induction, MDMs were infected with Sendai virus (final dilution = 1:200).
Total RNA from MDMs was isolated using the RNeasy Plus Mini Kit (QIAGEN) or NucleoSpin RNA Kit (Macherey-Nagel). Expression of mRNAs was determined in a 384-well format using QuantiTect SYBR Green RT-PCR Kit (QIAGEN) on an ABI7900 cycler (Applied Biosystems). Isoform-specific primers are listed in the Supplementary Methods.

Schott K., Fuchs N.V., Derua R., Mahboubi B., Schnellbächer E., Seifried J., Tondera C., Schmitz H., Shepard C., Brandariz-Nuñez A., Diaz-Griffero F., Reuter A., Kim B., Janssens V, & König R. (2018). Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55α holoenzymes during mitotic exit. Nature Communications, 9, 2227.

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Induction and Adoptive Transfer of EAU

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Six-week-old female and male C57BL/6 J, Anxa1^fl/fl or Anxa1^ΔLy6g mice were immunized with 500 μg of IRBP651-670 (Sangon Biotech) emulsified in complete Freund’s adjuvant (CFA; Sigma-Aldrich) and supplemented with 5.0 mg/mL Mycobacterium tuberculosis (MTB) strains. Intraperitoneal injection of 500 ng of pertussis toxin (List Biological) was performed at 0 h and 24 h. Splenic cells were collected from the mice at 1, 2, 3, and 4 weeks after immunization. We used mice without EAU induction as controls. The mice were housed in a dedicated pathogen-free facility maintained at 24 ± 2 °C under a 12 h light/12 h dark cycle. For IFN-α2a treatment, mice were given intraperitoneal injections of IFN-α2a (2,000 IU, Peprotech) daily for 2 weeks starting from day 14 after immunization. The clinical and histopathological scores of EAU were evaluated as described in the previous study^{81 (link)}. For adoptive transfer in murine model, freshly magnetically isolated and qualified neutrophils were resuspended in sterile PBS at a concentration of 1 × 10⁴ cells/µL, and 1 × 10⁶ cells were intravenously injected into the recipient mice through tail vein.

Wang Q., Ma J., Gong Y., Zhu L., Tang H., Ye X., Su G., Huang F., Tan S., Zuo X., Gao Y, & Yang P. (2024). Sex-specific circulating unconventional neutrophils determine immunological outcome of auto-inflammatory Behçet’s uveitis. Cell Discovery, 10, 47.

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Monocyte Priming and Activation Assay

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Cultures were performed in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10 % heat-inactivated FCS (HyClone, South Logan, UT), 4 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids, 100 U/ml penicillin, 100 ug/ml streptomycin (Life Technologies), 100 μg/ml streptomycin, and 50 μM 2-ME (MilliporeSigma, Rockville, MD). Monocytes were plated at 2 x 10⁵/0.1ml in 96-well plates (cytokine measurements) or 0.5 ml in 12-well plates (RNA analysis) and stimulated with an equal volume of untreated or MyB-treated tachyzoites (2x10⁶/ml), or with the TLR7/8 agonist R848 (300 ng/ml; InvivoGen, San Diego, CA). Priming with IFN-γ (10ng/ml) or IFN-α2a (10ng/ml) (PeproTech, Cranbury, NJ) was performed overnight, unless otherwise specified. To inhibit the mTORC1 pathway, Rapamycin (5nM) (10 (link)) or REDD-1 inducer (10μM) (11 (link)) (Millipore Sigma, Rockville, MD) were added to monocyte cultures 1 hour before stimulation with parasites or R848.

da Silva Amancio A.M., Mittereder L., Carletti A., Tosh K.W., Green D., Antonelli L.R., Gazzinelli R.T., Sher A, & Jankovic D. (2021). Interferons reset the differential capacity of human monocyte subsets to produce IL-12 in response to microbial stimulation. Journal of immunology (Baltimore, Md. : 1950), 206(7), 1642-1652.

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