What concentrations of plant extract (80% ethanolic extract) should be used in 96 well plates for a cytotoxicity study (MTT method, Hepg2 cell line)? What are the suitable controls?
Is there any literature available for this plat extract?? If yes , then you can take that LC50 value with 5 points above and below (diluted concentration) of that LC50 for cytotoxicity assay and If it is not reported, you can take any similar type of plant extract's LC50 value as a reference and take a range of concentrations (at least 10 dilution ).
You can take H2O2 / DMSO/detergent as a positive control and the solvent which will be used to dissolve your plant extract work as a Vehicle control.
Is there any literature available for this plat extract?? If yes , then you can take that LC50 value with 5 points above and below (diluted concentration) of that LC50 for cytotoxicity assay and If it is not reported, you can take any similar type of plant extract's LC50 value as a reference and take a range of concentrations (at least 10 dilution ).
You can take H2O2 / DMSO/detergent as a positive control and the solvent which will be used to dissolve your plant extract work as a Vehicle control.
If you can't find a starting concentration in the literature, then do a small pilot study: cover a several-log range from high nM to mM, depending on the solubility of whatever compounds may be of interest in the plant extract. Cover the widest range possible with 2-3 well/s concentration, understanding that this experiment is designed only to guide a more directed assay, and not to generate statistically meaningful results (and not for presentation). Also remember that what's soluble in 80% EtOH may precip out in, say, 1% EtOH when diluted into cell culture media or PBS or whatever your diluant will be. I don't know what levels of EtOH those HEP cells can tolerate, but you should definitely be able to find a study that treated those cells with some agent in EtOH vehicle, to get an idea of what a maximum reasonable concentration of EtOH in media will be. The literature will also guide treatment incubation time (i.e. 12-48 hrs?). Ideally you would treat several plates with identical doses, and harvest after different durations. This will not only give you a general dose-range to target, but also an ideal treatment duration. For an analytical experiment, try to treat the cells with log-spaced doses (i.e. 1, 3, 5, 10, 30 etc), unless you have good reason to select some other system. Remember- the closer the dose-spacing at the inflection points in a sigmoidal dose-response curve, the more precise your estimates of LC50 will be.
For MTT assays for cytotoxicity you have to standardize. Check some literature of your plant and compounds in it.
Hi Arbab,
You could follow certain references to decide the dose for the assay, however, activity and chemical composition of plant extracts varied as per the different geographical condition of plant collection, extraction procedure, solvent used and assay to be followed, therefore, previous references may not help you to decide the exact dose for the assay. For the assay you need to optimized the concentration of the extract, so I’ll suggest you to go for multiple doses starting from 10 µg to 500 µg (5-6 concentrations b/w this range). This will help you to determine the IC50 value of your sample as well. Later you can optimize the exact doses of the sample.
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