Recombinant human ifn α2

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Recombinant human IFN-α2 is a protein produced through recombinant DNA technology. It is a type I interferon that is involved in the immune response.

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IFN-α2 (6 citations)

6 protocols using recombinant human ifn α2

Phosphorylation of STAT Proteins in Whole Blood

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200 μl of whole blood was stained with anti-human CD4-BV510, anti-human CD8-APC-H7 and anti-human CD38-APC at previously determined optimum dilution for 15 mins at 37°C with or without recombinant human IFN-α2 (Biolegend) or recombinant human IFN-γ (ProSpec) at 100 ng/mL. Whole blood was lysed and fixed with Phosflow Lyse/fix 1x solution (BD Biosciences) and lymphocytes permeabilized with BD Perm III solution (BD Biosciences) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing anti-human pSTAT1-PECF594, anti-human pSTAT4-AF488 and anti-human pSTAT5-PE at previously determined optimum dilution for one hour at room temperature. Cells were washed and resuspended in staining buffer. Samples were acquired using a LSR Fortessa 4 (BD Biosciences) with Diva software, and analyzed using FlowJo software (version 6.0).

Burel J.G., Apte S.H., Groves P.L., Klein K., McCarthy J.S, & Doolan D.L. (2016). Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production. PLoS Pathogens, 12(9), e1005839.

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3D Pulmonary Microvessel Generation

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PMECs (Lonza) were used to generate 3D pulmonary microvessels. PMECs were cultured in EC medium from passages 6 to 8 using routine procedures. Three-dimensional, 150 μm diameter microvessels were fabricated based on published protocols (45 (link)). Microchannels were patterned in 7 mg/mL type I collagen (Corning) using nitinol wire, then cross-linked with 20 mM genipin (Wako). PMECs were suspended at 1 × 10⁶ cells/mL in EC medium and seeded into microchannels under static conditions for 30 minutes to promote cell adhesion. Microchannels were then perfused at 3 dyne/cm² for 48 hours in EC medium to form confluent pulmonary endothelium. To replicate the inflammatory state, microvessels were perfused with EC medium supplemented with 1000 U/mL recombinant human IFN-α2 (BioLegend) and 50 ng/mL recombinant human IFN-γ (R&D Systems) for 24 hours.

Casciola-Rosen L., Thiemann D.R., Andrade F., Trejo-Zambrano M.I., Leonard E.K., Spangler J.B., Skinner N.E., Bailey J., Yegnasubramanian S., Wang R., Vaghasia A.M., Gupta A., Cox A.L., Ray S.C., Linville R.M., Guo Z., Searson P.C., Machamer C.E., Desiderio S., Sauer L.M., Laeyendecker O., Garibaldi B.T., Gao L., Damarla M., Hassoun P.M., Hooper J.E., Mecoli C.A., Christopher-Stine L., Gutierrez-Alamillo L., Yang Q., Hines D., Clarke W.A., Rothman R.E., Pekosz A., Fenstermacher K.Z., Wang Z., Zeger S.L, & Rosen A. (2022). IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function. JCI Insight, 7(9), e158362.

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Isolation and Treatment of Amnion Epithelial Cells

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Isolation of primary human amnion cells were performed as described previously (Barrows et al., 2016 (link); Menon et al., 2013 (link)). Fetal membranes were obtained after placental delivery from women undergoing elective repeat cesarean section and uncomplicated pregnancies at term. The institutional review board (IRB) approval for discarded tissues was obtained prior to sample collection. Fetal membranes were dissected, and the amnion layer was peeled from choriodecidua, washed in warm saline, and small pieces (0.5 cm ²) were digested twice with trypsin and collagenase for 30 min at 37°C. The dig estion buffer was inactivated by DMEM complete media (DMEM/F12, Thermo Fisher Scientific) supplemented with 15% FBS and 1% P/S. The purity of the epithelial cells was determined by staining with cytokeratin (Pan-Cytokeratin, Abcam). Primary human amnion epithelial cells were treated with recombinant human IFN-λ1 (100 ng/ml, equal to 200–500 unit/ml, Biolegend) or recombinant human IFN-α2 (100 ng/ml, equal to 1.86×10⁴ units/ml, Biolegend) either 24 h prior to or following ZIKV infection at a MOI of 2.

Chen J., Liang Y., Yi P., Xu L., Hawkins H.K., Rossi S.L., Soong L., Cai J., Menon R, & Sun J. (2017). Outcomes of congenital Zika disease depend on timing of infection and maternal-fetal interferon action. Cell reports, 21(6), 1588-1599.

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Interferon-Induced Gene Expression Analysis

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HEK293, A549, and Calu-3 cells were incubated with different concentrations of recombinant human IFN-α2 (10³, 10⁴, and 10⁵ U/mL; BioLegend), recombinant human IFN-β (10³, 10⁴, and 10⁵ U/mL; PeproTech), recombinant human IFN-ω (10⁵, 10⁶, and 10⁷ U/mL; PeproTech), recombinant human IFN-γ (10³, 10⁴, and 10⁵ U/mL; BioLegend), and recombinant human IFN-λ2 (10³, 10⁴, and 10⁵ U/mL; PeproTech), respectively. At 24 h poststimulation, the cells were harvested, and the gene expression levels of α-SNAP, ISG56, IP-10, and MX1 were determined by qPCR.
A549 cells were incubated with IFN-α2 (10⁴ U/mL), IFN-β (10⁴ U/mL), IFN-γ (10⁴ U/mL), IFN-λ2 (10⁴ U/mL), and IFN-ω (10⁶ U/mL), respectively. At 12, 24, and 48 h poststimulation, the cells were harvested to determine α-SNAP mRNA levels by qPCR. The α-SNAP protein expression levels at 24 h poststimulation were determined by using flow cytometry.

Wang J., Luo J., Wen Z., Wang X., Shuai L., Zhong G., Wang C., Sun Z., Chen W., Ge J., Liu R., Wang X, & Bu Z. (2022). Alpha-Soluble NSF Attachment Protein Prevents the Cleavage of the SARS-CoV-2 Spike Protein by Functioning as an Interferon-Upregulated Furin Inhibitor. mBio, 13(1), e02443-21.

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Modulation of ZIKV infection by IFN-λ1 and IFN-α2 in Vero and JEG-3 cells

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Vero cells (African green monkey kidney epithelial cells) were maintained in D MEM (Thermo Fisher Scientific) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin. JEG-3 cells (a gift from Mariano A. Garcia-Blanco) were maintained in MEM (Thermo Fisher Scientific) supplemented with 10% FBS and penicillin/streptomycin. Cells were treated with recombinant human IFN-λ1 (100 ng/ml, equal to 200–500 unit/ml, Biolegend) or recombinant human IFN-α2 (100 ng/ml, equal to 1.86×10⁴ units/ml, Biolegend) either 24 h prior to or following ZIKV infection at a MOI of 2. The ZIKV strain (Asian lineage FSS13025) was provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA, Galveston, TX). ZIKV stocks were propagated in Vero cells and detected by titration.

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Cytokine and Inhibitor Treatment Protocol for Cellular Responses

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For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.
For BET inhibition, cells were treated with 1 µM JQ1 (Sigma) or 1 µM I-BET151 (GSK1210151A; Selleckchem). For the inhibition of pan-caspases, Z-VAD-FMK (A1902-25; Apexbio) was used at a final concentration of 20 µM. For sensitization to TRAIL-induced cell death, cells were treated with 1µM BV6 (S7597; Selleckchem). All drugs were dissolved in DMSO; DMSO was used as vehicle control.

Di Benedetto C., Khan T., Serrano-Saenz S., Rodriguez-Lemus A., Klomsiri C., Beutel T.M., Thach A., Walczak H, & Betancur P. (2023). Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis. Cancers, 15(3), 967.

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