Hey all
I want to see the affect of a certain drug on my cell line (cancer cell line).
I want to see how it alters the expression of a certain nuclear protein.
Since my cells are fast growing and protein synthesis usually takes 72 hours in mammalian cells, how should I seed and give treatment to the cells?
Should I seed them in low cell density and give treatment after overnight incubation? So that the there will be enough space left for cells to grow and divide until 72 hours?
Should I use media devoid of serum to prevent cells overgrowing the flask?
thanks and best
Hello V H Krishna Prasad
You should not devoid your cells of nutrients. The cells must grow happily under normal condition if you must see the actual effect of the drug on the cells. If you use media devoid of serum for 72 hours, you will be starving the cells because of which the cells will be under stress, and you may not obtain the expected result.
I would suggest that you perform a pilot experiment with different cell densities and then select the optimum seeding density that gives you 80% confluency at 72 hours. Seed the cells at the selected optimum seeding density, and after an overnight incubation, you may treat the cells. At this particular seeding density, you will know that the cells will have enough space to grow until 72 hours. You should always use media containing serum during the treatment period.
Wouldn’t using a 6-well plate or a petri dish (35mm or 60mm or 100mm) be better than the flask?
Best.
Thank you very much for your reply dear Malcolm Nobre .
I am giving treatment in triplicates (3 flasks for one concentration). I did use 6 well plate before, how ever protein yield was very low around 1.5 micro gram.
As you suggested a pilot study to understand at what cell density they will reach confluency for 72 hours is indeed required.
Thank you again dear Malcolm Nobre
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