Optimization of some parametric values of MTT for the determination of human melanoma (SK-Mel-30) cell viability

Abstract

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) assay is a widely used assessment method for the determination of anticancerogenic effects of active compounds including plant secondary metabolites. Recently, some important plant active ingredients have been widely investigated for anticancerogenic properties on melanoma cancer lines which are the most lethal type of skin cancer. Although some methods including DNA assay, 3H-thymidine incorporation and flow cytometry have been recommended for counting cells in the culture, MTT is one of the most frequent method and therefore, MTT assay needs to be optimized for melanoma cell lines. In this study, the MTT analytical procedure for determination of cell viability of human melanoma cell line (SK-Mel-30) was divided into nine steps and various parameters in each step (reagent amount, incubation time, centrifugation, solvent type, waiting time before spectrophotometric analysis and spectrophotometric parameters) were optimized. Optimum amount of MTT reagent and incubation time after MTT addition were determined as 10 µL and 4 h for 96 well plate, respectively. Various solvents were evaluated for solubility effectiveness of the formed crystals and DMSO was found to be the best solvent to dissolve the crystals. Waiting time before spectrophotometric reading and Uv-vis spectrums were also evaluated. At the end of the study a flowchart, presented the best analytical conditions, was constructed. Obtained findings can be used for the determination of anticancerogenic properties of plant ingredients.

Keywords

• Cell viability
• Melanoma
• MTT
• Optimization
• Validation.

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