Could someone suggest a highly sensitive, cost effective and efficient assay for estimating the secreted cytokines in body fluids or culture media. ELISA can be one alternative, but i have heard there are certain other colorimetric methods which are more Sensitive and accurate. Any leads, please?
Multiplex arrays are your answer. have a look at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1534009/ and as an aexample: http://131.220.28.201/wordpress/wp-content/uploads/2010/07/BD-Biosciences-FLEX.pdf. BD biosciences and Millipore do these, either using the Luminex or FACS systems with beads. Search for Multiplex and Luminex and you will get more hits than you read!
Thank you Michael and Rodrigo!
Yes, i have heard of luminex assay and most likely i am going to zero down on that.
Some time back we had a cytokine assay with randox for our Human CSF sample but we found the resultant little unconvincing and disappointing. Nonetheless, will try the luminex assay, as i have heard quite many positive reviews about it.
Since my sample is a rat sample this time, i am afraid i wont be able to use milteny biotec assay. But nonetheless, wonderful information. Thanks again for all the help.
RayBiotech has mouse arrays. I don't know about rat. They use very small sample volumes and the method is extremely sensitive.
I use the luminex and have had great results with biorad kits. Also Millipore has some kits but it all depends on your needs. The volume of fluid needed usually ranges between 20 and 50 ul.
Tissues will be homogenized in ice-cold Tris buffer (pH 7.2, 4 °C) containing 50 mM Tris, 1 mM EDTA, 6 mM MgCl2 and 5% (w/v) protease inhibitor cocktail (Sigma). After homogenization, samples will be sonicated for 10 s using an ultrasonic processor (Heat systems-Ultrasonic inc.) at a setting of 20 duty cycle, and then centrifuged at 20,800 g for 20 min at 4 °C in 3K300 centrifuges. Supernatants will be collected and cytokines were estimated using ELISA kits (R&D Systems) and reading will be taken in Elisa plate reader (Bio-Tek Instruments, Inc). Cytokine levels is expressed as pg of cytokines/mg protein ( Deak et al., 2003).
Hi Poojashri,
If you can use Flow Cytometry, then Cytometric Bead Arrays are the way to go by flow cytometry. At iQ Biosciences, we routinely perform cytokine release assays using in-vitro generated supernatants for functional & safety purposes. The benefits in using the CBA methods are significant (validated kit, perfect for limited sample volumes, multiple cytokines in ~ 4 hours).
The caveat to the CBA method is that you will need a flow cytometer, to purchase the kit, and software from BD Pharmingen in order to perform this. If you think you will perform this regularly, it is worth the investment if you already have a flow cytometer which is expensive. Otherwise, you may simply wish to work with contract research organizations that can perform this for you at a reasonable cost with fast turn around time.
I've pasted in some links to give you more background information as well as what representative data you may obtain as well. If you do decide to proceed with the CBA, please email me at [email protected] and I can share with you a refined protocol that we use here at iQ Biosciences internally. It reduces the volume of the reaction and still gives you solid results while saving you 50% on cost, etc.
Good Luck,
Omar
http://www.iqbiosciences.com/bioservices/safety_assessment/cytokine-release-assay/
https://www.bdbiosciences.com/research/cytometricbeadarray/
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