PK13 cells were transfected with either the empty pCAGGS plasmid or plasmids encoding various viral proteins as detailed in specific experiments. Empty pCAGGS was used as negative control for IFN antagonism, where PIV5-V-HA protein was included as positive control. At 24 hours post-transfection, cells were treated with 1000 U/ml of human IFN β (PBL). Following 24 hours of IFN β treatment, cells were infected with NDV-GFP as described previously (Park et al., 2003 (link)). Fluorescence images were obtained at 14 hours post-infection. In parallel experiments, VERO cells were first treated with 1000 U/ml of human IFN β (PBL) for 20 hours, then the cells were β transfected with either the empty pCAGGS plasmid or LPMV-V-HA plasmid. 24 hours post transfection the cells were infected with NDV-GFP described previously (Park et al., 2003 (link)). Fluorescence images were taken at 14 hours post-infection.
Human ifn β
Manufactured by PBL Assay Science
Sourced in United States
The Human IFN-β is a lab equipment product that functions as a recombinant human interferon beta. It is used in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse ifn β, by PBL Assay Science (5 mentions)
Human il 6, by BD (2 mentions)
Mouse il 6, by BD (2 mentions)
Ficoll paque premium, by GE Healthcare (2 mentions)
Citrate phosphate dextrose solution, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Human il 27, by Thermo Fisher Scientific (1 mentions)
Human il 6rα, by R&D Systems (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Human rantes, by R&D Systems (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
2 c methylcytidine 2cmc, by Merck Group (1 mentions)
Mouse ifn α, by PBL Assay Science (1 mentions)
Mouse tnf α, by BD (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Biostack4, by Agilent Technologies (1 mentions)
Tumor necrosis factor (tnf), by R&D Systems (1 mentions)
Lymphoprep density gradient medium, by STEMCELL (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
18 protocols using human ifn β
1
IFN Antagonism by Viral Proteins
2
Cytokine signaling in immune cells
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Human (#407306) and mouse (#407320) IFN-γ, human IL-6 (#407652) were purchased from Merck Millipore, human IFN-β (#11415–1) was from PBL Assay Science. Human IL-6Rα was from R&D Systems (#227-58-025), human IL-27 from Peprotech (#200–38). Other treatments used in this work include LPS (#L7895, Sigma) and ratjadone (#553590, Merck Millipore). Unless stated otherwise, IFN treatment was for one hour with 50 U/ml IFN-γ or 500 U/ml IFN-β in growth medium. Similarly, unless stated otherwise, IL-27 treatment was performed for 40 min at a concentration of 100 ng/ml. IL-6/IL-6R cotreatments also lasted for 40 min at concentrations of 200 and 250 ng/ml, respectively.
3
Cytokine ELISA Assay Protocol
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Commercial cytokine enzyme-linked immunosorbent assay (ELISA) kits used in this study were human IFN-β (PBL Assay Science) and human RANTES (R&D Systems). Cytokine levels in the media of cultured cells were determined according to the manufacturer's instructions. Absorbance was determined with FLUOstar Omega (BMG Labtech).
4
Inhibiting Human Norovirus Replication
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2′‐C‐Methylcytidine (2′CMC), an in vitro inhibitor of HNoV replication, was supplied by Sigma-Aldrich. Human IFN-β was purchased from PBL Assay Science and used at a concentration of 1,000 U/mL. IFN-neutralizing antibodies were obtained from PBL Assay Sciences and were used at the following concentrations: 1:4,000 for anti-IFN-α, 1:4,000 for anti-IFN-β, 1:4,000 for anti-IFN-β2, and 1:1,000 for anti-IFN-αβR2. HNoV GII.4 virus-like particles (VLPs) were purchased from The Native Antigen Company, and poly(I·C) was obtained from InvivoGen (catalog no. tlrl-picwlv). GII.4 Sydney and GII.6 HNoV-positive stool samples were kindly provided by J. Vinje (Centers for Disease Control and Prevention, USA) and S. M. Karst (University of Florida, USA), respectively. Stool samples were diluted with phosphate-buffered saline (PBS) to make a 10% (wt/vol) stock solution. Diluted stool samples were vortexed and centrifuged at 20,000 × g for 1 min. The supernatants were used for infection.
5
Cytokine Production Analysis by ELISA
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ELISA was used to detect the production of pro-inflammatory cytokines and type I interferon from cells. After infection, treatment and transfection of stimulants, cell supernatant was collected and analyzed cytokine production levels. Mouse IFN-α (PBL interferon source), mouse IFN-β (PBL interferon source), mouse IL-6 (BD biosciences) and mouse TNF-α (BD biosciences), human IFN-β (PBL interferon source) and human IL-6 (BD biosciences) were used for analysis according to manufacturer’s protocol.
6
SARS-CoV-2 ORF7a Overexpression and IFN-β Response
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HEK 293T-hACE2 cells (0.1 × 106) were seeded into a 0.01% poly-L-lysine solution-coated 24-well plate. Next day, cells were transfected with pCDNA SARS-CoV-2 ORF7a-V5/His (WT/H47Y; 1 µg) or empty vector plasmids. Twenty-four hours later, cells were treated with 250 units/mL of human IFN-β (PBL assay science, Piscataway, NJ, USA) or mock-treated (PBS). At sixteen hours posttreatment, cells were either mock-infected or infected with SARS-CoV-2-eGFP/ΔORF7a at 0.01 MOI. Cells were harvested 24 hpi followed by RNA isolation, cDNA synthesis and RT-qPCR, as described above (see SARS-CoV-2 infection of Calu-3 cells section). Cells from the duplicate wells were lysed and processed for immunoblotting for confirming SARS-CoV-2 ORF7a (WT/H47Y) expression (see Immunoblotting section).
7
IFN-I Luciferase Assay for SARS-CoV-2 ORF7a
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The IFN-I luciferase reporter assay was performed as described previously with some modifications [15 (link)]. Briefly, HEK 293T cells were seeded in a 12-well plate at a cell density of 0.25 × 106 cells/well. The next day, cells were cotransfected with pISG56-Luc (100 ng), pRL-CMV (5 ng), pCDNA SARS-CoV-2 ORF7a-V5/His (WT/H47Y; 2 µg) or empty vector plasmids. At 16 h posttransfection, cells were treated with 1000 units/mL of human IFN-β (PBL assay science) or mock-treated (PBS). At eight hours posttreatment, the cells were assayed for dual-luciferase activities using a Dual-Glo Luciferase Assay System (Promega) and a Biostack4 (BioTek) luminometer. Cells from the duplicate wells of mock-treatment conditions were lysed and processed for immunoblotting for confirming SARS-CoV-2 ORF7a (WT/H47Y) expression (see Immunoblotting section).
8
Isolation and Stimulation of Human Mononuclear Cells
9
Isolation and Stimulation of Human Mononuclear Cells
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Peripheral blood samples from adults were obtained from healthy volunteers at the National Institutes of Health (NIH) after informed consent with approval from the International Review Board (IRB) of the NIH (NIAID Protocol number 03-I-0263) and provided de-identified. Non-identifiable umbilical cord blood samples were obtained with the approval of the IRB of the Walter Reed National Military Medical Center (WRNMMC) (Protocol number WRNMMC-EDO-2018–0203). Umbilical cord blood was collected after delivery of the placenta into citrate-phosphate-dextrose solution (Sigma). Human blood was diluted in an equal volume of PBS and layered over Ficoll-Paque PREMIUM (GE Healthcare, Silver Spring, MD). MCs were isolated by centrifuging at 400×g for 30 minutes. MCs were washed twice and frozen in FCS/10% DMSO. On day of stimulation, MCs were thawed, washed, and rested in 48-well plates for 2 hours. Human IFNβ (PBL Assay Science) or R848 (InvivoGen, San Diego, CA), was then added for stimulation at 37°C for 16–18 hours prior to analysis by flow cytometry.
10
Cytokine Stimulation of Human PBMCs
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The study was approved by Institutional Review Boards at Yale University (following Yale melanoma skin SPORE institutional review board protocol). Healthy donors consented to donation of peripheral blood for research use.
Human PBMCs were isolated using Lymphoprep density gradient medium (STEMCELL). PBMCs were plated at 1 million cells per ml and stimulated with 1,000 U ml–1 human IFN-α2 (R&D systems), 1,000 U ml–1 human IFN-β (PBL Assay Science 11415), 1,000 U ml–1 human IFN-γ (PBL Assay Science), 1 µg ml–1 human IFN-III/IL-29 (R&D Systems), 100 ng ml–1 human IL-6 (NCI Biological Resources Branch Preclinical Biologics Repository), 20 ng ml–1 human TNF (R&D Systems), and combinatorial cytokines IFN-β + IL-6, IFN-β + TNF, IFN-β + IFN-γ at indicated concentrations above for up to 48 h.
Human PBMCs were isolated using Lymphoprep density gradient medium (STEMCELL). PBMCs were plated at 1 million cells per ml and stimulated with 1,000 U ml–1 human IFN-α2 (R&D systems), 1,000 U ml–1 human IFN-β (PBL Assay Science 11415), 1,000 U ml–1 human IFN-γ (PBL Assay Science), 1 µg ml–1 human IFN-III/IL-29 (R&D Systems), 100 ng ml–1 human IL-6 (NCI Biological Resources Branch Preclinical Biologics Repository), 20 ng ml–1 human TNF (R&D Systems), and combinatorial cytokines IFN-β + IL-6, IFN-β + TNF, IFN-β + IFN-γ at indicated concentrations above for up to 48 h.
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