HEK-293T cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (GenStar) and antibiotics. Vero E6 cells and A549 cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Transfection of cells was performed using Lipofectamine 3000 (Life Technologies) in Opti-MEM (Life Technologies) according to the manufacturer’s instructions. Cells were treated with IFN-α2a (catalog no. 11101–2; PBL Assay Science) at 1000 units/ml, MG132 (MCE) at 20 μM, Z-VAD-FMK (APE x BIO) at 20 μM, and NH4Cl (sigma-Aldrich) at 10 mM.
Ifn α2a
Manufactured by PBL Assay Science
Sourced in Germany, United States
IFN-α2a is a recombinant human interferon alpha-2a protein. It is a type I interferon that demonstrates antiviral, antiproliferative, and immunomodulatory activities.
Automatically generated - may contain errors
Lab products found in correlation
Poly 1 c, by InvivoGen (2 mentions)
Ifn β1a, by PBL Assay Science (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GenStar (1 mentions)
Z vad fmk, by Apexbio (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Ifn β, by PBL Assay Science (1 mentions)
Prism version 7, by GraphPad (1 mentions)
Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Immunotools (1 mentions)
Mouse monoclonal flag antibody, by Merck Group (1 mentions)
Tbk1 antibody, by Abcam (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Phospho tbk1, by Cell Signaling Technology (1 mentions)
Phospho irf3 antibody, by Cell Signaling Technology (1 mentions)
Irf3 antibody, by Santa Cruz Biotechnology (1 mentions)
Herring testis dna, by Merck Group (1 mentions)
14 protocols using ifn α2a
1
Cell Culture and Transfection Methods
2
SHIV Replication Assay with IFNα-2a
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Replication of SHIVs was accessed as described previously [25 (link)]. Briefly, 1x106 Ptm lymphocytes were infected at an MOI of 0.02 by spinoculation at 1200 x g for 90 minutes at room temperature. After spinoculation, cells were washed 4x with 1 ml of complete IMDM, re-suspended in 1.2 ml of complete IMDM and plated in two wells of a 48-well plate. Five hours after the initial infection, IFNα-2a (PBL Assay Science) was added to one well at a final concentration of 1,000 U/ml. Every three days, two-third of the cultures were harvested and cell-free supernatant were separated by pelleting at 650 x g for five minutes at room temperature. Cultures were replenished with fresh, complete IMDM, including with IFNα-2a if appropriate. SIV p27 concentrations were determined using a SIV p27 antigen ELISA (Advanced BioScience Laboratories). The data and statistical analyses were performed using Prism version 6.0c (GraphPad Software).
3
IFN-I Effect on ZIKV Replication
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To measure the impact of IFN-I treatment on ZIKV replication, 8 × 104 A549, Jeg3, SNB-19, or SH-SY5Y cells were either left untreated or pretreated with 1000 U/mL of IFNα-2a or IFNβ (PBL Assay Science, carrier-free) for 24 h in each well of a 24-well plate. After pretreatment, cells were infected at an MOI of 1 in a final volume of 250 µL of serum-free RPMI for 4–6 h. The inoculum was then aspirated and replenished with 1 mL of complete RPMI without IFN-I or containing 1000 U/mL of IFNα-2a or IFNβ. At 48 h post-infection (hpi), 250 uL of supernatants were harvested and cleared of cellular debris at 4 °C at 300G for 10 min and 2 × 100 uL aliquots were stored at −80 °C until titration by TCID50 assay. For the data analysis, all values were plotted, and statistical analyses performed using Prism version 7 (GraphPad Software, San Diego, CA, USA). Percent Relative Infection was determined by dividing the TCID50 titer in the IFNα- or IFNβ-treated sample by the untreated sample.
4
Macrophage Polarization and Stimulation
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One day following polarization of macrophages into M(-), M(IL-4), and M(HAGG+IL-1β), polarization media was washed off, and cells were treated with 20 ng/mL IFN-γ, 20 ng/mL IL-10, 1 μg/mL LPS (MD Biosciences), 1 μg/mL R848 (InvivoGen), 1000 U/mL IFN-α2a (PBL Assay Science), 10 μg/mL Poly (I:C) (InvivoGen), or 10 μg/mL ODN2395 CpG (InvivoGen) overnight. On the following day, macrophages were harvested using enzyme-free cell dissociation buffer (ThermoFisher).
5
Macrophage Activation by IFNs
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Macrophages were differentiated in macrophage medium (see above; with 4% human AB serum). After differentiation, the cells were treated with 10 ng ml−1 IFN-α-2a (PBL Assay Science; catalog no. 11100-1) or IFN-γ (ImmunoTools; catalog no. 11343534) for 48 h, stained with the 31 validation antibodies, and fixed with 2% PFA.
6
Characterization of cGAS Signaling Pathway
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The rabbit polyclonal antibody against cGAS was from Cell Signaling Technology and Sigma-Aldrich. The polyclonal antibody against RNF185 was from Abcam. The antibodies against hemagglutinin (HA), Myc, GFP, and ubiquitin were purchased from Santa Cruz Biotechnology. Mouse monoclonal Flag antibody and β-actin antibody were obtained from Sigma-Aldrich. The TBK1 antibody was from Abcam. The IRF3 antibody was from Santa Cruz Biotechnology. Phospho-TBK1 and Phospho-IRF3 antibody was from Cell Signaling Technology. The antibodies against CoxIV, Calreticulin and Calnexin were from Abcam. Anti-Flag (M2)-agarose was from Sigma-Aldrich.
Herring testis (HT) DNA was from Sigma. Salmon sperm DNA was from TREVIGEN. Poly(I:C) was purchased from Invivogen. IFNα2a was from PBL Assay Science. cGAMP was obtained from InvivoGen. In some experiments, cGAMP was delivered into cultured cells by digitonin permeabilization method as previously described [62 (link)].
Herring testis (HT) DNA was from Sigma. Salmon sperm DNA was from TREVIGEN. Poly(I:C) was purchased from Invivogen. IFNα2a was from PBL Assay Science. cGAMP was obtained from InvivoGen. In some experiments, cGAMP was delivered into cultured cells by digitonin permeabilization method as previously described [62 (link)].
7
CD1d Expression on Immune Cells
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CD1d expression on the cell surface of peripheral cells, including T cells, B cells, NK cells, and monocytes from healthy individuals was analyzed by flow cytometry. Monocytes were identified through FSC and SSC gating and expression of CD14, while T (CD3+), B (CD19+), and NK (CD3–/CD14–/CD19– and CD56+) cells reside in a lymphocyte gate comprising cells with less granularity and size. Furthermore, peripheral blood cells were treated with or without 100 IU/ml IFN-α2a or 1000 IU/ml IFN-λ3 (both PBL Assay Science) over 24 hours, and CD1d expression was assessed on monocytes defined through CD14 and CD16.
8
Evaluating EGCG and IFN-α2a in Hepatitis C Virus Assays
9
Measuring IFN-neutralizing Capacity
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The IFN-neutralizing capacity of S95021 and benchmark antibodies was tested with the help of reporter HEK-BlueTM IFN-α/β cells (InvivoGen) that express alkaline phosphatase (AP) under the inducible ISG54 promoter after ISGF binding to the IFN-stimulated response elements (ISRE) in the promoter as previously reported [33 (link)]. The cells were grown in DMEM (Naxo), containing 10% heat inactivated FBS and supplemented with 30 μg/mL blasticidin (InvivoGen) and 100 μg/mL Zeocin (InvivoGen). IFN-α2a was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany) and all other subtypes were from PBL Assay Science and used at the following final concentrations: IFN-α2a 200 U/ml, IFN-α1 50 U/ml, IFN-α4, IFN-α5, IFN-α7 and IFN-α21 25 U/ml, IFN-α10 and IFN-α17 12.5 U/ml, IFN-α8 6.25 U/ml, IFN-α6 3 U/ml, IFN-α16 1.5 U/ml and IFN-α14 0.6 U/ml. Cells were stimulated with optimized concentrations of type I IFNs that were preincubated for 2 h with serial dilutions of monoclonal antibodies. QUANTI-Blue TM (InvivoGen) colorimetric enzyme assay was used to determine AP in the cell culture supernatants after 21 h of incubation. OD was measured at 620 nm with a Multiscan MCC/340 (Labsystems) ELISA reader. IC50 values were calculated from the dose-response curves.
10
HTNV Infection of HUVEC and A549 Cells
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HUVEC cells (ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in ECM BulletKit (ScienCell Research Laboratories, Carlsbad, CA, USA) in a 5% CO2 incubator. A549 cells (ATCC Cat# CRM-CCL-185, RRID:CVCL_0023) were grown in our laboratory in DMEM with 10% FBS (Thermo Scientific, Waltham, MA, USA) in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76–118 was cultured in Vero E6 cells (ATCC Cat# CRL-1586, RRID:CVCL_0574) in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein (NP) as previously described (22 (link)). The TCID50 was 105/ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source (Piscataway, NJ, USA) and dissolved in the buffer provided by the manufacturer (composition not disclosed). HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.
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