via HTS I found Inhibitors (IC50 between 1 to 5uM) for a protein that is involved in a specific pathway. Now I am doing cell-based assays to see the In-vivo effect.
I, Incubate the cpds with the cells for 20 hrs. For the assay, I remove the media (containing the cpds) and add a specific media (doesn't has the cpds) followed by 6hrs of incubation. Stain and image the cells.
Within the 6hrs the effect be reduced or the cpds be lost out of the cells (due to dilution)?
Will the effect be more prominent if I have cpds in constant contact with the cells?
I am using MEF´s
Removing the compounds and adding fresh media without compound and incubating for a further 6 hours could diminish the effect of the compound, thus allowing the cells to recover and preventing you seeing the effect after staining.
So adding compound to the 'specific' media also should prevent this from happening. Why do you replace with media without the compound?
Thanks Arvind.
You mean I should add the cpds in the specific media?
That specific media is the part of the assay.
There is no exact reason as to why not to include the cpds in it. I thought 20hrs should be good enough for the cpds to be incorporated and start showing the effect in the cellular metabolism. So didn't include it while doing the assay (specific media).
For some cpds, I do see an effect.
But today, I thought that maybe the effect could be more pronounced if I had it in contact all the time. Especially since they are not tight binders
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