Mouse IFN beta kit HTRF®

Name: Mouse IFN beta kit
Brand: Cisbio
SKU: 44620
Availability: InStock

Associated products

The HTRF mouse IFN beta kit is designed for the quantification of mouse IFN beta release in cell supernatant.

No-wash
Low sample consumption
Under 2h bench time

Download the protocols

The HTRF mouse IFN beta kit is designed for the quantification of mouse IFN beta release in cell supernatant.

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Overview

Type I interferons, interferon alpha (IFN-α) and interferon beta (IFN-β), play essential roles in the innate immune response to viral infections. IFN-β is produced by fibroblasts, macrophages, and dendritic cells, as well as endothelial cells. The expression of the IFN-β gene is controlled by transcription factors like IRF-3, c-Jun/ATF-2, and NF-kappaB downstream of Pattern Recognition Receptors, for example Toll-like receptors. In particular, IFN-β is the primary functional readout of cGas-cGAMP-STING pathway activation. Once secreted, IFN-β binds and activates the IFNAR1/IFNAR2 receptor complex, which relies on STAT1/2 transcription factors. Phosphorylated STAT1/2 leads to the induction of anti-viral gene expression.

Benefits

SCALABLE FROM 96 TO 1536 WELLS
PANEL QUANTIFICATION

Assay principle

Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Cisbio low-volume 96-well plate in 20 µL). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run up to a 1536-well format by simply resizing each addition volume proportionally.

Assay data analysis

The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases.

To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page.

Cisbio has also worked with Myassays.com to help you with your data analysis. By clicking on this link, you’ll be able to access a free online software to run your IFN beta analysis.

Technical specifications of mouse IFN beta kit

Sample size	16 µL
Final assay volume	20 µL
Kit components	Lyophilized standard, frozen detection antibodies, buffers, & protocol
LOD & LOQ (in Diluent)	4 pg/mL & 17 pg/mL
Range	17 - 2,000 pg/mL
Time to result	Overnight at RT
Species	Mouse only

Intra and inter assay

Intra assay (n=24)

Sample	Mean [IFN-β] (pg/mL)	CV
1	75	10%
2	200	3%
3	1125	3%
	Mean CV	5.3%

Inter assay (n=6)

Sample	Mean [IFN-β] (pg/mL)	CV
1	58	3.6%
2	348	3.3%
3	1092	7.3%
	Mean CV	4.7%

Spike and recovery

A fixed concentration of recombinant mouse IFN-beta was spiked into several cell supernatants containing known concentrations of mouse IFN-beta. Samples were mixed at a 1:1 volume ratio. The set of responses obtained from the standard curve was compared to the calculated expected values.

Dilutional linearity

A cell supernatant containing a known concentration of mouse IFN-beta was serially diluted in the culture media used to generate the sample. The set of responses obtained from the mouse IFN-beta standard curve was compared to the calculated expected values.

The mouse STING-specific agonists CMA and DMXAA trigger IFN-β release from NIH-3T3 fibroblast cells

The mouse NIH-3T3 fibroblast cell line was plated in a 96-well culture plate (100,000 cells/well) and incubated for 6h. The cells were then treated with 200 µM of the synthetic mouse STING-specific agonists CMA and DMXAA (under 100 µL). After 24h incubation, cell supernatants were collected and secreted levels of IFN-beta were measured following the procedure given in the HTRF kit’s package insert.

Mouse IFN-beta kit_NIH-3T3 cells treated with CMA and DMXAA

IFN-β secretion by the murine RAW 264.7 macrophage cell line treated with several STING agonists

The murine RAW 264.7 macrophage cell line was plated in a 96-well culture plate (100,000 cells/well) and incubated overnight. The cells were then treated with a fixed concentration of the synthetic STING agonists CMA, DMXAA, and ADUS100, as well as with the endogenous STING agonist 2'3'-cGAMP (under 100 µL). After 24h secretion, cell supernatants were collected and levels of released IFN-beta were measured following the procedure given in the HTRF kit’s package insert.

HTRF mouse IFN-beta kit_RAW 264.7 cells treated with several STING agonists

Dose-dependent secretion of IFN-β by the murine RAW 264.7 macrophage cell line treated with 2’3’-cGAMP and ADUS100

The murine RAW 264.7 macrophage cell line was plated in a 96-well culture plate (100,000 cells/well) and incubated for 5h. The cells were then treated with increasing concentrations of either the endogenous STING agonist 2'3'-cGAMP, or the synthetic small molecule ADUS100 (under 100 µL). After an overnight secretion, cell supernatants were collected and levels of released IFN-beta were measured following the procedure given in the HTRF kit’s package insert.

HTRF mouse IFN-beta kit_RAW 264.7 cells treated with 2’3’-cGAMP and ADUS100

How to measure multiple cytokines from 1 sample

What to expect at the bench - Videos

How to freeze PBMC

Guideline for PBMC freezing - Technical Notes

Guidelines from PBMC isolation to cytokine assay optimisation

Perform and optimize cytokine assays on PBMC - Technical Notes

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One sample to measure them all - Posters

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In collaboration with Blood assay solution - Application Notes

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HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

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Cytokine assays: a guide to success with HTRF

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Portfolio of Life Sciences Cytokine Assays

This leaflet presents the analytical performances of each assay - Brochures

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Get the brochure about technology comparison. - Brochures

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