Human IL-6R Reporter Assay Kit

SIZE	SKU	PRICE
1 x-96 well format assays	IB53001	—
3 x-32 assays in-96 well format	IB53001-32	—

SIZE	SKU
3 x-32 assays in-96 well format	IB53001-32
1 x-96 well format assays	IB53001

Add to quote

Specifications
Data
Target Background
Documentation
Overview
Citation
Related Products

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Interleukin-6 Receptor (IL-6R). INDIGO’s IL-6R reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of IL-6R. In addition to IL-6R Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human IL-6R. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

Clear, Reproducible Results
All-Inclusive Assay Systems
Exceptional Cell Viability Post-Thaw
Consistent Results Lot to Lot

Product Specifications

Target Type		Cytokine Receptor
Species		Human
Kit Components		IL-6R Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) IL-6, 600 ng/mL Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Shipping Requirements		Dry Ice
Storage temperature		-80C

Data

IL-6R Inhibition assays. 200 ul / well of IL-6R Reporter Cell suspension was dispensed into 96-well assay plates, which were then incubated for 4 hours. For inhibition-mode assays, prior to the end of the pre-culture period, CSM was supplemented with ~ 125 pg IL-6/ml (an approximate EC80 concentration). The IL-6-supplemented CSM was then used to prepare the various treatment media. The anti-IL-6R antibody Tocilizumab (Sino Biological) was diluted using IL-6-supplemented CSM to produce final assay concentrations beginning at 50 ug/mL and proceeding with serial 10-fold decrements. Pre-culture media were discarded and 200 ul/well of the prepared treatment media were dispensed (n = 5/conc.), including ‘vehicle only’ control wells. After 22 hr incubation treatment media were discarded, Luciferase Detection Reagent was added, and RLU/well were quantified. Plots are RLU vs. Log10[ng/mL] for the various test materials. Error bars depict +/- %CV.

IL-6R Agonist dose-response assays. IL-6R activation assays were performed according to the protocol provided in this Technical Manual. 200 ul / well of IL-6R Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The reference peptide IL-6 (provided; ProspecBio) was diluted using CSM to produce treatment media at the desired assay concentrations. The pre-culture media were discarded from the assay wells and 200 ul per well of respective treatment media were dispensed (n = 4/conc.). Following a 22-hour incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of % CV and Z’ were determined. Non-linear regression analyses and EC50 calculations were performed using GraphPad Prism software. Plots show Fold Activation vs. Log10[pg/mL] for the various treatment materials; error bars depict +/- %CV.

Target Background

This assay utilizes proprietary human cells that provide constitutive expression of the Human Interleukin-6 Receptor (IL-6R).

Interleukin-6 (IL-6) is considered one of the most prominent pro-inflammatory cytokines and causative agent of many autoimmune disorders. Chronic overproduction of IL-6 has been associated with wide-ranging illnesses such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), Gastric and Prostate Cancers, Alzheimer’s disease, Atherosclerosis, Cardiovascular disease and Stroke. In addition, the ‘cytokine storm’ caused by viral infections such as COVID have led to severe adverse outcomes. Considering its significant role and involvement in the progression of many types of disease, IL-6 and its receptor IL-6R continues to command much interest as a target for the development of novel, specific, and predominantly inhibitory drugs and antibodies. For example, blockade of IL-6R by the neutralizing monoclonal antibody tocilizumab has been approved in more than 100 countries for the treatment of rheumatoid arthritis.

IL-6 binds to a membrane-bound IL-6 receptor (IL-6R), and the complex of IL-6 and IL-6R associates with a second receptor protein, glycoprotein 130 kDa (gp130), which dimerizes and initiates signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT). Phosphorylation and activation of the transcription factor STAT3 is the signaling mechanism exploited by the reporter cells included in this kit.