Hi everyone,
Do you have any experience in preparing sample dilutions to measure the concentrations of inflammatory cytokines (TNFα, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, and so forth) for an ELISA when it comes to any rat brain tissue, especially hippocampus. I'd prefer not to perform a pretest, and waste any well-strips beforehand. Based on your experience from different labs, what would be the expected protein concentration ranges of these cytokines in a supernatant saved from tissue homogenate prepared from a healthy rat (Sprague Dawley) brain tissue (hippocampus)?
Kind regards,
Dear Gokhan Gokhaner
I suggest the following articles:
1. https://www.cancer.iu.edu/research-trials/facilities/mac/documents/bioplexarticle-rat-kit-lysis-conditions.pdf
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801052/
3. Article Rat pro-inflammatory cytokine and cytokine related mRNA real...
4. https://www.nature.com/articles/s41598-019-50708-w?proof=t
5. https://www.frontiersin.org/articles/10.3389/fphar.2020.562084/full
Best Regards
Dr. Fatemeh Khozaei
Halo Gokhan,
I understand that you do not want to use part of your kits doing pilot studies as kits are expensive, but this is the best way to discover the best dilution factor for each specific antibody. There are many variables that could be different between your samples and other authors' samples, so I would not assume that risk going ahead with the whole protocol
Dear Dr. Fatemeh Khozaei
Thank you for your detailed reply. I appreciate that. It's been quite helpful for the last couple of days. It actually gave me some insight.
Dear Dr. Cristian Pérez-Fernández
I understand that I need to perform a pretest just to make sure about the protein concentrations in the samples. Based on the detection range of the kit, now, I consider performing a pretest using a much narrower dilution range.
Dear Mr Gökhaner I am really interested in the results you obtained because I am currently in the same situation you were, trying yo measure ILs by ELISA in hippocampus mice homogenates. So, could you tell me which dilution factor would you consider optimal?
Thank you very much
Hi Professor Taoro, The papers and documents that I've been advised to check are on this very thread. They have been posted by Fatemeh Khozaei on this thread. You can easily find them by looking at previous replies. This paper (Hulse et al 2004), which has been posted by Fatemeh Khozaei especially seems to be worth to look at and have a read. Yet again as far as I know, it is the rat tissue but not mice tissue that was studied in the paper. Hulse, R. E., Kunkler, P. E., Fedynyshyn, J. P., & Kraig, R. P. (2004). Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue. Journal of neuroscience methods, 136(1), 87–98. https://doi.org/10.1016/j.jneumeth.2003.12.023 On a note, I have not been able to measure the cytokine levels, as reactive materials, kit components, and other disposables have not arrived at our university yet. Nice to hear that your dilution rate worked! I might even use that dilution for measurement! Kind regards, Gokhan
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