Determining Optimal Cell Density and Culture Medium Volume simultaneously in MTT Cell Proliferation Assay for Adherent Cancer Cell Lines

Author Name: Mohammadreza Moradi, Reza Solgi, Rezvan Najafi, Hamid Tanzadehpanah, *Massoud Saidijam
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Introduction: The MTT assay without optimization of the factors affecting cell metabolism can lead to false reports of the tested drugs. In this study, cell density and cell culture medium volume were optimized in a 96-well plate for Hepa1-6 cells as an adherent cell line. Methods: Hepa1-6 cells were seeded in the two different volumes, five cell densities, and three incubation times. After the incubation times, the optical densities were read at 570nm. The absorbances of wells in 100 and 150µl of cell culture medium volumes were compared and the optimum cell density was funded through the mean absorbance analysis of wells in the optimum volume. Results: The Independent sample t-test showed a significant difference (p-value=0.01) between the absorbances of wells with 100 and 150 µl of volumes. Also, Pearson’s correlation coefficient indicated that the optimum cell culture medium was 150 µl. The optimum cell density was 5000 cells/well obtained from the liner portion of the standard curve related to the optimum volume. Discussion: The optimal cell density and volume of cell culture medium for hepa1-6 cell line were found. In addition, a simple practical protocol was suggested to find the optimal factors mentioned for the adherent cell line in MTT assay.


The drug-based investigations for cancer treatment are initiated via a quick screening of candidate antitumor agents against tumor cell lines in vitro. Tumor cell viability is determined following the exposure of the mentioned cell lines to some drugs for time duration. To detect viability of the living cells through mitochondrial dehydrogenase activities, MTT assay is as the best known enzyme-based and most versatile quantitative method explained by Massom in 1983(Mosmann, 1983), which is also reliable for testing cytotoxicity and cell proliferation. It has been widely used with various modifications in recent years (Yang, et al., 2015). To generate the reduced equivalents, such as NADH and NADPH, yellow MTT tetrazolium salt (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is partly decreased by metabolically active cells with the help of dehydrogenase enzymes. Solubilization and quantification of the resultant intracellular purple formazan can be done through spectrophotometry.

However, false high or low cell counts may be calculated due to the significant modification of MTT-specific activity caused by some factors and various parameters affecting cellular metabolism. To prevent nutrient depletion, such parameters as cell density, fresh or old culture medium volumes, concentration, and exposure time should be incorporated into MTT. Thus, to minimize confusing effects, establishment of suitably controlling parameters for each cell line in the assay is of high importance (Sylvester, 2011). Among all, the two most important parameters are cell density and culture medium volume. If a high count of cells is seeded or less pure medium culture than proper is selected, a shortage of nutrients and non-drug-related death will occur and a false report of the tested drug will be resulted. To obtain a reliable result in MTT assay, any factors affecting the metabolic activities of cells for each cell line should be optimized. This is the first study to optimize MTT assay via a simultaneous focus on cell density and culture medium volume. According to the findings of this study, an easy and practical protocol could be proposed for determining the two mentioned parameters in the MTT cell proliferation assay for adherent cell lines. The result of this study can be applied to adherent cancer cell lines.


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