IFN-I cytokine levels were measured by ELISA in RAW 264.7 cells and by using an IFN reporter cell in THP-1 cells. RAW 264.7 cells were seeded in 96-well plate at a density of 5 × 104 cells/well and incubated overnight for experiment. Svg3 was transfected into cells using Lipofectamine reagent at a final concentration of 25 nM and incubated at 37°C and 5% CO2 for 24 h. The medium was collected to test the concentration of IFN-β. THP-1 cells were seeded at densities of 1 × 104 cells/well in a 96-well plate. Cells were treated with Svg3 or cGAMP, respectively, for 24 h. At the same time, HEK-Blue IFN-α/β cells (InvivoGen) were seeded at densities of 1 × 104 cells/well in a 96-well plate. Then THP-1 cell supernatant was added to HEK-Blue IFN-α/β cells for 24 h. Cell supernatant (50 μL)was collected from each sample and added to 150 μL of QUANTI-Blue SEAP detection medium (InvivoGen) and incubated for 2 h at 37°C. SEAP activity was assessed by measuring the absorbance at 630 nm on a plate reader.
Hek blue ifn α β cells
Manufactured by InvivoGen
Sourced in United States
HEK-Blue IFN-α/β cells are a reporter cell line derived from human embryonic kidney (HEK) 293 cells. They stably express an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an interferon (IFN)-inducible promoter. The cells are designed to detect the presence of type I interferons (IFN-α and IFN-β) in the cell culture supernatant.
Automatically generated - may contain errors
Lab products found in correlation
Zeocin, by InvivoGen (4 mentions)
Blasticidin, by InvivoGen (4 mentions)
Quanti blue, by InvivoGen (4 mentions)
Normocin, by InvivoGen (3 mentions)
B16 blue ifn α β cells, by InvivoGen (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Quanti blue seap detection medium, by InvivoGen (2 mentions)
Ficoll hypaque density gradient, by Harvard Bioscience (2 mentions)
3 deoxyguanosine 5 triphosphate, by TriLink (2 mentions)
Set of 4 on targetplus pde12 sirna lq 017946 01 0002, by Horizon Discovery (2 mentions)
Set of 4 on targetplus enpp1 sirna lq 003809, by Horizon Discovery (2 mentions)
Anti pde12 antibody ab87738, by Abcam (2 mentions)
Hsv 60 lyovec, by InvivoGen (2 mentions)
Recombinant enpp1, by R&D Systems (2 mentions)
Trpsin, by Merck Group (2 mentions)
Anti enpp1 antibody, by Cell Signaling Technology (2 mentions)
Herring testes dna, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Collagenase d, by Roche (2 mentions)
33 protocols using hek blue ifn α β cells
1
IFN-I Cytokine Quantification in Cell Lines
2
Quantifying Cytokine and Interferon Release
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IL-1β and IFNβ release was measured in cell supernatants by ELISA (DY201, DY814, R&D Systems) according to the manufacturer’s instructions. A bioassay for type I interferons was performed using HEK Blue IFN-α/β cells (Invivogen). HEK Blue IFN-α/β cells were stimulated for 20 h with supernatant from SeV infected THP1 cells and SEAP activity in the supernatant was measured by Quantiblue solution (rep-qbs, InvivoGen) according to the manufacturer’s conditions.
3
Quantification of IFN-I Cytokine Levels
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IFN-I cytokine levels were measured by ELISA in RAW264.7 cells and by using a IFN reporter cell in THP-1 cells. RAW264.7 cells were seeded in 96-well plate at density of 5X104 cells/well and incubated overnight for experiment. Svg3 was transfected into cells using lipofectamine reagent at a final concentration of 25 nM and incubated at 37 °C and 5% CO2 for 24 h. The medium was collected to test the concentration of IFN-β. THP-1 cells were seeded at densities of 1×104 cells/well in a 96-well plate. Cells were treated with Svg3 or cGAMP respectively for 24 h. At the same time, HEK-Blue™ IFN-α/β cells (Invivogen) were seeded at densities of 1×104 cells/well in a 96-well plate. Then THP-1 cell supernatant was added to HEK-Blue™ IFNα/β cells for 24 h. 50 μL cell supernatant was collected from each sample and added to 150 μL of QUANTI-Blue SEAP detection medium (InvivoGen) and incubated for 2 h at 37 °C. SEAP activity was assessed by measuring the absorbance at 630 nm on a plate reader.
4
Quantifying IFN-β Secretion via cGAMP Stimulation
5
Culturing HEK293 and Immune Cell Lines
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HEK293T and HEK293FT cells were maintained in Dulbecco's High Glucose Modified Eagle's Medium (DMEM, Hyclone) containing 10% heat-inactivated fetal bovine serum (FBS, Biological Industries), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco). HEK-Blue IFN-α/β cells (InvivoGen) were propagated in DMEM with 10% FBS, 100 μg/mL normocin, 30 μg/mL blasticidin and 100 μg/mL zeocin (InvivoGen). THP1, Jurkat, Raji, and Ramos cells were cultured in Roswell Park Memorial Institute (RPMI, Hyclone) 1640 supplemented with 10% FBS. THP1 monocytes were differentiated into adherent macrophages by 48 h incubation with 200 nM phorbol 12-myristate 13-acetate (PMA, P1585) followed by 48 h rest in PMA-free medium. All cells were maintained at 37 °C in a humidified incubator with 5% CO2.
6
Type I IFN Measurement Protocol
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Bioactive type I IFN was measured on cell supernatants by use of HEK-Blue™ IFN-α/β cells as reporter cells according to the manufacturer instructions (Invivogen). Type I IFN was measured using a cell-based assay. Briefly, L929 cells were cultured in DMEM 5%FCS and seeded into 96 well plates. Supernatant from stimulated BMDC’s were added as serial dilutions to the cells for 24h at 37°C alongside a positive control of muIFNα (100IU/ml). The next day, cells were infected with VSV/V10 and the cells were incubated until plaques were visible under the microscope. A 50% protection of virus-induced cell death in a well was used to define 1 U/ml of Type I IFN.
7
Masking and Unmasking IFN Signaling
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Example 5
In this example, it is shown that the masked IFN fusion protein (anti-CD138-IFNα2) does not activate the type I IFN signaling pathway. The activation could then be restored following Matriptase ST 14 treatment. Briefly, using HEK-Blue™ IFN-α/β cells (Invivogen, San Diego, Calif.) activation of the type I IFN signaling pathway is detected using a reporter gene expressing secreted embryonic alkaline phosphatase (SEAP) with gene activation monitored by quantitating alkaline phosphatase activity in the supernatant of treated cell. HEK-Blue™ IFN-α/β cells were incubated with hIFNα2, anti-CD138 masked IFNα2 w/o MST14, anti-CD138 masked IFNα2 w MST14; and an anti-CD138-IFNα2 for twenty-four (24) hours. The results show IFNα activity in masked anti-CD138-IFNα2 was found to be reduced to less than 10% of that seen in the unmasked protein; IFNα2 activity was restored to nearly 100% following matriptase/ST14 treatment (See,
8
Cytokine Quantification in Cell Culture
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TNFα and IL-6 were measured in culture supernatants with ELISA (Human TNF-alpha DuoSet; R&D systems and Human Interleukin 6 Cytoset kit; Invitrogen, Thermo Fisher, Scientific). Type I IFN bioactivity was measured in culture supernatants by a reporter assay system (HEK-Blue™ IFN-α/β cells; In vivoGen) according to the manufacturer’s instructions. Recombinant human IFNβ1a was used for calibration.
9
Measuring Bioactive Human Type I IFN
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Bioactive human type I IFN was measured on cell supernatants using HEK‐Blue™ IFN‐α/β cells as reporter cells according to the manufacturer instructions (InvivoGen) and as described by the manufacturer.
10
Measuring IFN-I Bioactivity in Plasma
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Protocols for the measurement of IFN-I bioactivity using a cell-based reporter system were adapted from a previous study (29 (link)). HEK-Blue IFN-α/β cells were purchased from InvivoGen (San Diego, CA, USA). In this study, the bioactivity of IFN-I in plasma was measured.
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