Hey all
I have seeded cancer cells in 6 well plate and intend to check the expression of certain protein with and without treatment with the drug at our disposal.
The cells have adhered and reached around 60-70% confluency by yesterday.
Should I give them treatment for 12 hours or 24 hours or just 2 hours? The drug has an IC50 of 5 micro molar for this cell line.
Also how do I justify the treatment time I provided for them. And how is this going to change the expression of protein ?
Thank you and best
Hello Krishna,
The time/duration of drug treatment for any cell types is depend on your experimental aim or question you are looking to answer, irrespective of cell type/drug. If you are looking or want to test a functional consequences (activity or phosphorylation like p-ERK or G-protein activities) then few minutes to 1/2 hours of treatment is sufficient. If you want evaluate the effect of any drug on the expression or regulations of particular protein (from transcription to translation) by xyz drug, then I suggest you to treat your cells for 72h, because most protein needs around 72h to synthesize from DNA-mRNA-protein. Hope this might helpful. Good luck with your experiments.
Thank you very much for your reply Sabbir Khan . In case of the treatment I give, some of the cells loose their potential to adhere over time. So must I centrifuge the cells with the treatment media to recover them ? Or should I discard the treatment media with the floating cells and add fresh media and scrape the attached cells for protein extraction?
Thank you again Sabbir Khan
V H Krishna Prasad you can collect the floating cells from the media, mostly they dead, especially if you are looking for cell death (apoptosis) by Annexin-V staining via flow cytometry. No need for other assays like western blotting or qPCR. Make sure, the drug treatment does not affect the cell attachment (adhesion protein), where relatively healthy cells might float in media.
Sabbir
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