Mouse IL-6 ELISA Kit from Life Technologies

January 22, 2013

University of Illinois at Urbana-Champaign
Food Science and Human Nutrition
Postdoctoral Research Associate

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Company:

Life Technologies

Product Name:

Mouse IL-6 ELISA kit

Catalog Number:

KMC0062

ImageInflammation is a part of a host response to infection but excessive inflammation is also related to a number of chronic diseases including cancer and cardiovascular disease. Interleukin-6 (IL-6) is a multifunctional cytokine associated with the immune response, inflammation, apoptosis and cell survival. It is associated during hepatic acute phase inflammation, inflammatory-related carcinogenesis and atherosclerotic plaque and instability. The goal of my experiments were to measure the production of IL-6 in LPS-stimulated and non-stimulated macrophages.  The protocol of this kit was simple and easy to follow. Supernatants from LPS-stimulated and non-stimulated macrophages were diluted with standard diluent and plated in the provided ELISA plate. The total incubation time is short and the assay can be done in one day.

Experimental Design and Results Summary

Application

ELISA

Starting Material

Macrophages were allowed to grow to at least 90% confluency in a 6-well plate, after which, the supernatant was removed and cells were treated with LPS for 24 h. After 24 h treatment, supernatant was collected from LPS- and non-stimulated macrophages.

Protocol Overview

Supernatant was diluted with standard diluent buffer and plated on the ELISA plate provided for 2 h together with the provided IL-6 standard (prepared at different concentrations). After washing and incubation with biotin conjugate mouse IL-6, streptavidin-HRP and stabilized chromogen, the reaction was stopped by adding stop solution and absorbance was read at 450nm.

Tips

The protocol is very simple and easy to follow; the only caveat is that you need to wash the well multiple times in between incubations. Also, it is slightly hard to follow when to stop the reaction after adding the stabilized chromogen though the kit said that it should be between 20-30 minutes of incubation.

Results Summary

Overall, the mouse IL-6 ELISA kit performed very well. The standard curve generated from the different concentrations of mouse IL-6 generated an R2 of 0.9921. Also, the difference in the production of IL-6 between LPS-stimulated and non-stimulated macrophages is very significant and well-differentiated (see image below).

Additional Notes

Since the dilution of the collected supernatant is very important for the success of the experiment as its absorbance readings need to fall in the standard curve, it would be very helpful if the kit provided an idea of what dilution to use for a particular amount of cells seeded and supernatant used rather than suggesting that samples producing signals greater than that of the highest standard should be reanalyzed. This will help researchers, especially the new ones, in saving their time and effort from looking up literature in order to find what dilution to use and going back to calculate the appropriate dilution. Or worse, you have to redo the assay all over again and spend time and money again. Also, it would be nice if they could provide an additional bottle of standard diluent buffer as some samples may need to be diluted more.

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Summary

The Good

The kit is simple and easy to follow protocol.

The Bad

The researchers should know how to play with dilutions and too many washings are needed.

The Bottom Line

A powerful ELISA kit for measuring IL-6 in cell culture supernatants, worthy of the price.

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