Control and experimental (EZH2-knockdown or EZH2 inhibitor-treated) cells were propagated until they reached 70–80% confluency. The culture media were replaced 24–48 hours before collection of the replenished media. Type I interferon cytokines were analyzed using human IFN alpha DuoSet ELISA and human IFN beta DuoSet ELISA kits (R&D systems) according to the manufacturer’s instructions, and the absorbance was measured using Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).
Human ifn beta duoset elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human IFN-beta DuoSet ELISA kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed for the quantitative measurement of human interferon beta (IFN-beta) in cell culture supernatants, serum, and plasma.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Magnetic protein a g beads, by Thermo Fisher Scientific (2 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Hcytmag 60k px38, by Merck Group (1 mentions)
Glomax microplate reader, by Promega (1 mentions)
7 protocols using human ifn beta duoset elisa kit
1
Quantifying Type I Interferon Levels
2
Quantifying Human IFN-β by ELISA
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For the quantitative determination of human IFN-β concentrations in supernatants of cultured cells, ELISA assays using commercially available Human IFN-beta DuoSet ELISA kits (R&D Systems, France) were performed according to the manufacturer’s instructions.
3
Western Blot Analysis of vIL-6 and IFNβ
4
Quantifying IFNβ1 Silencing in Cancer Cells
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ELISA was used to quantify the amounts of IFNβ1 in the supernatant of cancer cells to validate the IFNβ1 silencing by the interference RNA. MCF7 was seeded in full growth media in a six‐well plate at a confluency of 2 × 105. The next day, siIFNβ1 plus transfection mix was added to the cells and incubated for 6 h. Transfection media was aspirated and reduced serum media with 10 µg·mL−1 of Poly(I:C) (InvivoGen, CA, USA) was added to the cells for 3 days. Secreted IFNβ1 was detected with human IFN‐beta DuoSet ELISA kit (R&D systems, Minneapolis, MN, USA) according to manufacturer’s instructions. Optical density was measured at 450 nm using GloMax microplate reader (Promega GmbH). A standard curve was generated using the absorbance values of the standards and used to calculate the concentration of IFNβ1 in each sample.
5
Western Blot Analysis of vIL-6 and IFNβ
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Whole cell lysates were prepared in SDS-sample buffer (60mM Tris.HCl pH6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and separated by SDS-PAGE and then transferred to nitrocellulose membranes (GE Healthcare). The membrane was blocked in PBS-T (PBS with 0.1% Tween20) with 5% nonfat milk (Apex) and then incubated with the indicated primary antibody at 4°C overnight. Please refer to the KEY RESOURCES TABLE for antibodies used in this study. The vIL-6 antibody was purified from the supernatant of v6m 12.1.1 hybridomas (ATCC) using magnetic Protein A/G beads (Thermo Fisher). IFNβ protein in the supernatant were quantified by human IFN-beta DuoSet ELISA kit (R&D Systems) according to the manufacturer’s protocol. IFNβ protein levels (pg/ml) were calculated based on the standard curve generated in the assay.
6
Cytokine Profiling in BKV-Infected Renal Cells
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To test IFN and additional cytokine production, culture supernatants from mock and BKV infected RPTE and VEC at various time points were collected and stored at -80°C until processing. IFNβ levels were determined with the Human IFN-beta DuoSet ELISA kit (R&D Systems) following manufacturer’s instructions. A 38-plex human cytokine Luminex panel (HCYTMAG-60K-PX38, Millipore Sigma) was used to assay secretion of 38 additional cytokines. The collected supernatants were submitted to the Hillman Cancer Center (UHCC) Luminex Core Laboratory affiliated with the University of Pittsburgh Medical Center for the procedures.
7
Quantifying IFNb1 Silencing in Cancer Cells
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ELISA was used to quantify the amounts of IFNb1 in the supernatant of cancer cells to validate the IFNb1 silencing by the interference RNA. MCF7 was seeded in full growth media in a 6-well plate at a confluency of 2 x 10 5 . The next day, siIFNb1 plus transfection mix was added to the cells and incubated for 6 hours.
Transfection media was aspirated and reduced serum media with 10 µg/mL of Poly(I:C) (InvivoGen, California, USA) was added to the cells for three days. Secreted IFNb1 was detected with human IFN-beta DuoSet ELISA kit (R&D systems, Minnesota, USA) according to manufacturer's instructions. Optical density was measured at 450 nm using GloMax microplate reader (Promega GmbH, Walldorf, Germany). A standard curve was generated using the absorbance values of the standards and used to calculate the concentration of IFNb1 in each sample.
Gene set enrichment analysis. Gene set enrichment analysis was carried out using GSEA v.4.0.3 51, 52 . Comparison between groups (CAF1_CC-U vs. CAF1_CC-P; MCF7_MC-U vs.
MCF7_MC-E/P) was done using gene sets extracted from GSEA. All parameters were kept as default. Nominal P values were calculated based on random genes permutations.
Transfection media was aspirated and reduced serum media with 10 µg/mL of Poly(I:C) (InvivoGen, California, USA) was added to the cells for three days. Secreted IFNb1 was detected with human IFN-beta DuoSet ELISA kit (R&D systems, Minnesota, USA) according to manufacturer's instructions. Optical density was measured at 450 nm using GloMax microplate reader (Promega GmbH, Walldorf, Germany). A standard curve was generated using the absorbance values of the standards and used to calculate the concentration of IFNb1 in each sample.
Gene set enrichment analysis. Gene set enrichment analysis was carried out using GSEA v.4.0.3 51, 52 . Comparison between groups (CAF1_CC-U vs. CAF1_CC-P; MCF7_MC-U vs.
MCF7_MC-E/P) was done using gene sets extracted from GSEA. All parameters were kept as default. Nominal P values were calculated based on random genes permutations.
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