The clinical isolate PF-25013-18 (PF13) of ZIKV has been previously described [10 (link)]. Cell lines used in this study included the A549-Dual™cell line (InvivoGen, a549d-nfis), referred to hereafter as “A549 cells”, and the HEK-Blue™ IFN-α/β cell line (InvivoGen, San Diego, CA, USA) which possesses the HEK-293A backbone and is referred to hereafter as “HEK-293A cells”. Both cell lines were cultured at 37 °C with 5% CO2 in MEM Eagle medium, supplemented with 10% heat inactivated fetal bovine serum and 2 mmol·L−1l -Glutamine, 1 mmol·L−1 sodium pyruvate, 100 U·mL−1 of penicillin, 0.1 mg·mL−1 of streptomycin and 0.5 µg·mL−1 of fungizone (PAN Biotech, Aidenbach, Germany). Additionally, A549 cellular growth medium was supplemented with 10 µg·mL−1 blasticidin and 100 μg·mL−1 zeocin (InvivoGen) and HEK-293A cell growth medium was supplemented with 30 µg·mL−1 blasticidin and 100 μg·mL−1 zeocin (InvivoGen). Cells were harvested and stored as frozen pellets for further protein or mRNA analysis.
Hek blue ifn α β cell line
Manufactured by InvivoGen
Sourced in United States
The HEK-Blue IFN-α/β cell line is a reporter cell line derived from human embryonic kidney (HEK) 293 cells. It is designed to detect the activity of type I interferons, including interferon-alpha (IFN-α) and interferon-beta (IFN-β), by monitoring the activation of the ISRE (interferon-stimulated response element) signaling pathway.
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Lab products found in correlation
Zeocin, by InvivoGen (2 mentions)
Blasticidin, by InvivoGen (2 mentions)
Recombinant human ifn β standard, by InvivoGen (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fungizone, by PAN Biotech (1 mentions)
Penicillin, by PAN Biotech (1 mentions)
Streptomycin, by PAN Biotech (1 mentions)
Quanti blue detection reagent, by InvivoGen (1 mentions)
Tryptose phosphate broth, by Thermo Fisher Scientific (1 mentions)
Heat inactivated fbs, by Lonza (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Hek293t, by InvivoGen (1 mentions)
Bhk 21, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bafetinib, by Adooq (1 mentions)
Loxoribine, by InvivoGen (1 mentions)
Lumikine mifn α, by InvivoGen (1 mentions)
4 protocols using hek blue ifn α β cell line
1
Zika Virus ZIKV Infection Protocol
2
IFN-I Bioactivity Assay in Cell Culture
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Human IFN-I bioactivity in tissue culture supernatants was measured with reference to a recombinant human IFN-β standard (InvivoGen) using HEK-BlueIFN-α/β cell line (InvivoGen) and QUANTI-Blue detection reagent, following manufacturer’s instructions. In minireplicon assays, levels of bioactive IFN-I in tissue culture supernatants were determined by evaluating protection capacity against cytopathic effect (CPE) after 24h infection of Vero cells with VSV at moi = 0.1. As control, cells were treated with either 1000, 100 and 10 I.U./ml of IFN-I for 16 hours. Under these conditions, 10 I.U./ml protected against VSV induced CPE.
3
Cell Culture Conditions for Virus Research
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A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza). BHK-21 were also supplemented with 20% Tryptose Phosphate Broth (18050039, Thermo Scientific). HEK-293T cells were supplemented with sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM). HEK-Blue IFN-α/β cell line (InvivoGen) was maintained in HEK-293T media supplemented with 10μg/ml blasticidin (InvivoGen) and 200 μg/ml Zeocin (InvivoGen). Vero E6 cells (Cercopithecus aethiops kidney epithelial cells, ATCC CCL-81) were cultured in Minimum Eagle Medium (MEM) (11095–080, Gibco), supplemented with 2 mM L-glutamine, 50 U/mL penicillin-streptomycin and 7.5% heat-inactivated FBS. A549, BHK-21, HEK-293T and Vero E6 cell lines were originally provided by J.C. de la Torre (The Scripps Research Institute, La Jolla, CA) and HEK-Blue IFN-α/β cell line by S. Sharma (La Jolla Institute for Allergy and Immunology, La Jolla, CA)
4
Measuring Cytokine Responses in Immune Cells
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For cytokine measurement in culture supernatants, 1 × 106 total BMDCs, 5 × 104 FACS-purified pDCs, 1 × 105 CAL-1 or 3 × 106 PBMCs were left untreated or pre-treated for 1 h with the SFKs inhibitor PP2 (EMD Millipore), the inhibitor bafetinib (Adooq, Irvine, CA, USA) or the equivalent concentration of DMSO control, followed by 15 h in media in presence or absence of 0.1 μM CpG-B ODN 1668 (Roche), 1 μM CpG-A ODN 2216, 100 μM loxoribine (mouse cells) or 1 μg ml−1 R848 (human cells) (all from InvivoGen, San Diego, CA, USA).
Mouse type I IFN bioactivity was measured with reference to a recombinant mouse IFN-β standard (Research Diagnostics, Concord, MA, USA) using a L-929 cell line transfected with an interferon-sensitive luciferase. Human type I IFN bioactivity was measured with reference to a recombinant human IFN-β standard (InvivoGen) using HEK-Blue IFN-α/β cell line (InvivoGen). Mouse serum IFN-α, mouse and human TNF were measured, respectively, by luminescent ELISA (LumiKine mIFN-α, InvivoGen) or by ELISA (Mouse TNF-α ELISA Ready-SET-Go!, eBioscience, and Human TNF-α ELISA MAX, Biolegend) as described in the manufacturer's protocol.
Mouse type I IFN bioactivity was measured with reference to a recombinant mouse IFN-β standard (Research Diagnostics, Concord, MA, USA) using a L-929 cell line transfected with an interferon-sensitive luciferase. Human type I IFN bioactivity was measured with reference to a recombinant human IFN-β standard (InvivoGen) using HEK-Blue IFN-α/β cell line (InvivoGen). Mouse serum IFN-α, mouse and human TNF were measured, respectively, by luminescent ELISA (LumiKine mIFN-α, InvivoGen) or by ELISA (Mouse TNF-α ELISA Ready-SET-Go!, eBioscience, and Human TNF-α ELISA MAX, Biolegend) as described in the manufacturer's protocol.
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