MTT assay and IC50 calculation

The MTT assay is a widely used colorimetric technique that assesses cell viability and proliferation based on the metabolic activity of living cells. It operates on the principle that viable cells possess active mitochondrial dehydrogenase enzymes capable of reducing the yellow tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), into insoluble purple formazan crystals. These crystals accumulate within the cells and are directly proportional to the number of metabolically active cells present. Following incubation, a solubilizing agent such as DMSO is added to dissolve the formazan crystals, producing a colored solution. The intensity of this color, measured using a spectrophotometer at a wavelength of 570 nm, correlates with cell viability: higher absorbance signifies greater numbers of viable cells. This straightforward and reliable method is extensively applied in various research fields, including cell biology, pharmacology, and toxicology, to evaluate cell health, drug efficacy, and cytotoxicity. The MTT assay's ease of use, sensitivity, and reproducibility make it a valuable tool for a broad range of cellular studies.
The percentage of cell viability in an MTT assay is calculated by comparing the absorbance of treated samples to that of control (untreated) samples. First, the absorbance values are measured for each well, including blanks (containing only medium and reagents without cells) to account for background absorbance. The blank absorbance is subtracted from both the treated and control sample absorbances to obtain corrected values. The cell viability percentage is then determined by dividing the corrected absorbance of the treated samples by the corrected absorbance of the control samples, and multiplying by 100. This calculation yields the relative viability of the treated cells as a percentage of the control, providing a quantitative measure of how the treatment affects cell health and proliferation. The formula used is:
% Cell Viability=[(OD of Control Sample−OD of Blank)/(OD of Treated Sample−OD of Blank​)]×100
In the MTT assay, the "control" wells contain cells treated under standard conditions (e.g., untreated or treated with a vehicle), serving as a baseline for expected cell viability, while the "blank" wells contain only culture medium and MTT reagent, without cells, to account for background absorbance; these controls are crucial for accurately assessing the impact of experimental treatments on cell viability by providing reference points for comparison and correcting for non-cellular contributions to absorbance measurements.

[@Mightychondria2009]

This was so helpful. Thankyou sm✨

[@alvynakartikakhairunisa2417]

about the linear regression, why dont use the %viability value? why %cytotoxic? and how it can be 100-%viability?

[@marcolorful]

To make a direct correlation between the drug conc and the the effect: more drug --> more %cytotox, but there are better tools to calculate the IC50, because making it a linear regression is not correct.

[@ravispharmacologyandanatom6365]

Dear one... When we calculate the MTT assay for the cancer cell such as MCF-7 and MDA-MB-231, we want our drug to be cytotoxic to these cells. That time we can use% cytotoxicity. Contrary to this when you experiment is about neuroprotection % cell viability will be more accurate. Further 100% cell viability is a theoritical concept generally we consider control group where there is no treatment was used have 100% cell viability. It would be wise to do the cell count to exactly know the cell density. Then again it's just a quick method. We have other more accurate methods like FITC, where we can exactly see how much cell are alive and how much cells are undergone apoptosis. Please check out my latest research to know more about MTT and FITC.... doi.org/10.1111/cbdd.14458