I intend on testing my compound on different cancer cell lines to test whether they are cytotoxic or cytostatic. However, I am unsure of what concentration of compound I should use. If the concentration is too high, of course, it ends up killing all the cells. If the concentration is too low, cells are not killed making the drug look ineffective. Of course, using EC50 values could have been one of the ways, but comparing different cell lines with different EC50 values once again will be an unfair way of comparing drugs in my opinion. Any feedback on this will be appreciated.
Dear Zohaib
I agree with you that EC50 is not the best way but at least it gives you a clue about the dose to start with. Best wishes
In addition to determine EC50/IC50/LC50, You can looks on literature of your compounds or their derivatives/parent compounds to get an idea regarding the toxicity of your compounds.
Good Luck!
you can use Cytotoxicity assay by using the CytoTox 96 kit (Promega, UK) or any other kit
I would recommend some quick cheap assay to determine EC50 in a panel of cell lines. And then specific staining for apoptosis or other programmed cell death at EC50 to demonstrate the nature of cytotoxicity.
I like the idea of Farhadul to look for information on similar compounds regarding toxicity in vivo. However, often a single radical added can change the whole mechanism of drug action.
you have to generate full dose response curves for each compound/cell line, you can do this quite easily in 96-well format, use alamar blue to quantify cell survival at the end point. Seed cells in 90uL (say 2000-5000 cells/well) then add drug(s) as 10X conc the next day (10uL). Prep serial dilutions (in media) of your drug in a separate 96-well plate, eg from 100uM. then half-Log dilutions (1:3) all the way down to 10nM or less (8-10 point dose response). Ensure DMSO % is the same across all wells (eg 0.1%). Then add to your cells in triplicate - use a multichannel pipette. Final conc. of drugs will be 10uM to 1nM. Wait 2-3 days then assay for viability.
Normalise to the DMSO controls for each cell line (eg % survival). Only then can you compare to other cell lines. The shape of the DR curve tells you cytostatic response vs cytotoxic eg a cytotoxic response is usually has a steep hillslope, where % survival drops from say 100% to 0% in one dose step. Cytostatic curves flatten out at the higher doses and does not kill 100%
Quantifying as AUC (area under the curve) might help alongside your other data.
I would recommend you too use LDH cytotoxic assay kit. You can get the product info below and all other similar product can be used easily too.
https://www.dojindo.com/TechnicalManual/Manual_CK12.pdf
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