We want to evaluate cytotoxicity effect of a bacterial peptide on HeLa cells. Should we dilute the bacterial peptide in medium with serum (FBS) or medium without serum? When use medium with serum (FBS), we have two problems: 1. Control cells grow rapidly and a high OD is obtained. 2. The cytotoxicity of the peptide is not shown correctly and we have negative results (control cells and treated cells have same OD).
Hello
It does not make any difference if you dilute the bacterial peptide with medium containing FBS or without FBS because finally when you add the diluted compound to the wells containing cells, the cells are already in complete medium (with FBS).
To test the cytotoxic effect of any compound the cells have to be grown in their normal medium (with FBS) so that the cells do not undergo stress owing to depletion of nutrients in medium without FBS.
Just a suggestion. You would need to optimize on the concentration of the bacterial peptide.
I hope this would help.
Good Luck.
For MTT assay I culture epthelial cell line (for example A549) in complete medium with 10% FBS for 24 h (100 ul/well) and the next day I add 100 ul of cytotoxic compound in medium without FBS. So I reduce the final concentration of FBS twice. It should help to slow down the growth of cell line.
Furthermore, if control and peptide-treated cells grow at the same level, it propably means that the concentration of the peptide is too low to cause the cytotoxic effect.
I hope it helped :)
Unless you are having diverging results using complete versus incomplete medium, my guess is that FBS is not the problem.
Consider the following possibilities causing a high absorbance and no difference between treated and untreated cells:
1. Too many cells.
2. Too little peptide.
3. Too long incubation with MTT.
To better understand what is going on you can run a control with something that you know that will affect cell viability (e.g. 20% DMSO in medium) and see what happens.
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