I want to do an MTT assay, and I have eight concentrations of an extract on MCF-7 cells I arranged 96 well plates by adding (Negative control: cells+ media without adding the drug) and (Positive control: cells with the DMSO), then the eight concentrations, all with three replicate, is this right or not? any advice, please, thank you
Hello Areej,
The blank well in MTT assay will be required because it will represent the background absorbance value created by the medium itself that must be subtracted from the final values of the samples, and positive and negative controls.
FYI: The blank in the MTT assay is a cell-free medium plus an MTT solution and a solubilizing buffer.
The positive control well will contain untreated cells, MTT solution and a solubilizing buffer. Viability will be close to 100%. Positive control is used as a reference for your sample cell viability, it is always been set as 100% cell viability.
The negative control well will contain only dead cells, MTT solution and a solubilizing buffer.
Best.
Hello Areej,
The assay your are setting up is similar to a cytotoxicity assay where you are interested in checking the safety of the extracts with significant biological activity.
For this kind of assay, after adding your extract at 8 concentrations in the 96 well microtitre plate, the negative control is the most important because it is what will help you calculate the effect of your drug only on the cells (% inhibitions of drug). This can be done by substrating OD value you have at each concentration of the drug from the average OD of the negative control, divide the answer you have by the average OD of the negative control and finally multiply this answer by a hundred to have % inhibition values at each concentration.
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