I'm trying to screen for a suitable range of concentration of Gefitinib for my subsequent ELISA assay, the final aim is to select a Gefitinib concentration that does not kill cells+give off inflammatory response. I will detect the levels of inflammatory markers and compare it to the reading after certain treatment, which believe to be able to reduce the level of inflammatory markers.
I have tested on the range of concentration between 80-600 ng/mL and the result from my MTS assay did not show much significance in terms of cell cytotoxicity, meaning the viability is more or less the same (60%-90% viability compared to my vehicle control), and the trend looks odd (cell viability increases as drug concentration increases). I prepared my Gefitinib drug stock with 100% DMSO and dilute it with growth medium for all my assays. The initial range of concentration is determined based on literature mentioning the Cmax value of Gefitinib in healthy individuals, which I believe is more relevant to my study, since I'm treating normal human epidermal keratinocyte cells (NHEK) .
Anyone has any experience with treating NHEK cells with Gefitinib? I've seen a lot of studies being done with cancer cells but not with normal cells. I'm unsure of the limit of concentration to be used, of course at higher drug concentrations cells are going to be killed and inflammatory response will be more significant, but it does not make sense if I just use a high concentration without any reference to real life/clinical situations?
I'm asking this so that I can save resource (the drugs used are expensive, so as MTS reagent and ELISA kits) and not to blindly increase the concentration just for the sake of getting a positive result. I hope to get some enlightenment on this!
Hello Phylia Chew
Have you gone through the paper attached below?
The investigators have used 3, 10, 30nM concentrations of the drug (EGFRi) including Gefitinib prepared from a 10 mM stock solution dissolved in DMSO. Keratinocytes isolated from donated human tissue were used in the study. A higher drug concentration of 1μM has been found to be toxic to cells.
Article EGFR inhibitors switch keratinocytes from a proliferative to...
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