The IFN-I production assay was performed as described previously (Xia et al., 2018 (link)). Briefly, HEK293T cells (1 × 105 cells per well in a 24-well plate) were co-transfected with 10 ng of IFN-β promoter reporter plasmid, 4 ng of Renilla luciferase plasmid, 20-80 ng of viral protein expression plasmid using X-treme-GENE™ 9 transfection reagent with a ratio 1:2. Empty pXJ vector was used to ensure the same total amount (100 ng) of plasmids in each well. Cells were induced by co-transfection with 4 ng/well of stimulator expressing plasmids [(RIG-I (2CARD), MAVS, TBK1, IKKε, or IRF3)], same amount of empty pXJ vector was used as non-stimulated control. At 24 h post-transfection, the cells were assayed for dual-luciferase activities according to the manufacturer’s instructions (Promega). For the IFN-I signaling, HEK293T cells (1 × 105 cells/well, 24-well plate) were co-transfected with 250 ng of ISRE promoter reporter plasmid, 20 ng of Renilla luciferase plasmid, and 230 ng of viral protein expression plasmid. At 16 h post-transfection, the transfected cells were treated with 1,000 units/ml of human IFN-α (Millipore). After another 8 h incubation, the cells were lysed and performed dual-luciferase reporter assays according to the manufacturer’s instructions (Promega). Luciferase signals were read by Cytation 5 (Bio Tek, Winooski, VT).
Human ifnα
Manufactured by Merck Group
Human IFNα is a recombinant protein that corresponds to the alpha subunit of the human interferon protein. It is a laboratory reagent used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by Merck Group (2 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Dual luciferase activities, by Promega (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Affini pure f ab 2 fragment goat antihuman igm, by Jackson ImmunoResearch (1 mentions)
Cd19 microbeads, by Miltenyi Biotec (1 mentions)
Tlr7 ligand r848, by Enzo Life Sciences (1 mentions)
Ifn α, by Merck Group (1 mentions)
Ribavirin, by Merck Group (1 mentions)
Homoharringtonine, by Merck Group (1 mentions)
Doxazosin, by Merck Group (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti mtor, by Santa Cruz Biotechnology (1 mentions)
Anti phospho akt, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Nsc118218, by Selleck Chemicals (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti survivin, by Cell Signaling Technology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
7 protocols using human ifnα
1
IFN-I Production and Signaling Assay
2
Isolation and Stimulation of Human B Cells
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B cells were purified from PBMCs by labeling cells with CD19 microBeads and positively selecting CD19+ B cells (Miltenyi Biotec, Germany). The purity of B cells was always above 97%. For in vitro experiments, isolated human CD19+ B cells were cultured in RPMI 1640 medium containing 10% FBS and stimulated with TLR7 ligand R848 (1 μg/ml, Enzo Life Sciences International), TLR9 ligand CpG-2006S (0.3 μM, Invitrogen), AffiniPure F(ab’)2 Fragment Goat Anti-human IgM (10 μg/ml, Jackson ImmunoResearch Laboratories) or human IFN-α (1000 U/ml, Millipore). The study protocol was approved by the research ethics committee of Nanjing University.
3
Investigating CHIKV Infection in Vero Cells
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Vero cells (ATCC CCL-81) were seeded into 96 well plates and cultured for 24 hrs at the indicated temperatures and treated with human IFNα (Sigma I 2396; a mixture of 10 IFNα species) for 4 hrs. CHIKV was added (MOI = 0.05) and incubation continued at the same temperature. Cytopathic effect (CPE) was assessed by crystal violet staining after 3 days. For virus replication Vero cells after seeding at the indicated temperatures in triplicate in 24 well plates were infected with CHIKV for 6 hrs, were washed and then incubated at the same temperatures. Viral titers in the supernatants were assessed at the indicated times after washing.
4
Broad-Spectrum Antiviral Agent Preparation
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Ribavirin, MPA, brequanir, and homoharringtonine were purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). Human IFNα (Sigma-Aldrich, H6166) was dissolved in phosphate-buffered saline (PBS). Stock of JAK inhibitor 1 (SC-204021, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was dissolved in DMSO with a final concentration of 5 mg/ml. A library of 94 safe-in-human broad-spectrum antiviral agents (https://drugvirus.info ) was dissolved in DMSO with a stock concentration of 10 mM.
5
Molecular Mechanisms of Ovarian Cancer
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Human ovarian cancer cell lines (SKOV-3, 2774 and OVCAR-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human IFN-α and IFN-γ were obtained from Sigma (St. Louis, MO). The chemical doxazosin was also purchased from Sigma. The JAK1/2 kinase inhibitor INCB18424 (Ruxolitinib) and STAT1 inhibitor (NSC118218) were obtained from Selleck Chemicals (Houston, TX), and stock solutions were prepared in DMSO. NSC74859 (S31-201), a specific STAT3 inhibitor, was purchased from Calbiochem Chemicals (La Jolla, CA). The following primary antibodies were used in this study: anti-JAK1, anti-phospho-JAK1, anti-JAK2, anti-phospho-JAK2, anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3, anti-caspase-3, anti-cMyc, anti-Bcl-2, anti-Bax, anti-p53, anti-survivin, and anti-COX-2 (Cell Signaling, Beverly, MA), anti-PARP, anti-XIAP (BD Biosciences, San Jose, CA), anti-cyclin D1, anti-CDK4, anti-Akt, anti-phospho-Akt, anti-TYK2, anti-phospho-TYK2, anti-PI3K, anti-phospho-PI3K, anti-mTOR, anti-phospho-mTOR, anti-PKCδ, anti-phospho-PKCδ, anti-STAT2, anti-phospho-STAT2, anti-p70S6K, and anti-phospho-p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21, and anti-p27 (Oncogene, San Diego, CA).
6
NDV Infection Modulates STAT1 Activation
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To detect STAT1 during infection, A549 and Vero cells were advance-infected with NDVs at a MOI of 3 diluted with DMEM. For infection, cells were cultured in 6-well plates, washed 3 times with PBS, and incubated with NDVs in 600 μL DMEM per well at 37°C for 30 min. Supernatant was discarded and cells were cultured in DMEM containing 2% FBS (Gibco). At indicated time points post infection, cells were stimulated using 500 U/mL human IFN-α or IFN-γ (Sigma) in 1 mL DMEM at 37°C for 15 min. After stimulation, cells were lysed and subjected to Western blot for STAT1 and STAT2. Uninfected cells were stimulated with IFN and tested as negative controls.
To analyze the influence of viral proteins on infection and STAT1 phosphorylation, plasmids encoding P/V proteins or domains were transfected into cells ahead of infection. For transfection, cells cultured in 6-well plates were washed 3 times by PBS and transfected with plasmids or empty plasmid (4 μg/well) using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Supernatants were washed out and cells were cultured in DMEM containing 2% FBS (Gibco). At 48 h post transfection, cells were used for infection and IFN-stimulation as above-described.
To analyze the influence of viral proteins on infection and STAT1 phosphorylation, plasmids encoding P/V proteins or domains were transfected into cells ahead of infection. For transfection, cells cultured in 6-well plates were washed 3 times by PBS and transfected with plasmids or empty plasmid (4 μg/well) using Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Supernatants were washed out and cells were cultured in DMEM containing 2% FBS (Gibco). At 48 h post transfection, cells were used for infection and IFN-stimulation as above-described.
7
Monocyte Differentiation and Activation
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Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) of healthy, HIV‐negative donors using magnetic CD14 Microbeads (Miltenyi Biotech) and AutoMACS Pro Separator (Miltenyi Biotech) according to manufacturer instructions. 4 × 106 Monocytes were seeded in 2 ml RPMI (Gibco) supplemented with 10% heat‐inactivated FCS, 1% penicillin–streptomycin, 20 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF; PeproTech), and 20 ng/ml interleukin 4 (IL‐4; PeproTech). Two days after seeding, 2 ml of fresh media were added to cells in differentiation. Cells were allowed to differentiate for 6 days before starting the experiments. Where indicated, 50 ng/ml of LPS from E. coli (Sigma Aldrich) or 0.3 unit/μl of human IFNα (Sigma Aldrich) were added.
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