EVALUATION OF CYTOTOXICITY POTENTIAL OF TEST SAMPLE USING DIMETHYLTHIAZOL-DIPHENYLTETRAZOLIUM BROMIDE (MTT) ASSAY

                                                                  STUDY PROTOCOL

1.0 INTRODUCTION:

The MTT (dimethylthiazol-diphenyltetrazolium bromide) colorimetric assay is based on mitochondrial uptake and succinate dehydrogenase reduction of soluble, yellow, MTT tetrazolium salt to an insoluble blue MTT formazan product. Cytotoxicity assays were among the first in vitro bioassay methods used to predict the toxicity of substances to various tissues. In vitro cytotoxicity testing provides a crucial means for safety assessment and screening, and for ranking compounds.

2.0 METHODOLOGY:

· Cell culture

· Mouse fibroblast L929 cell line will be procured from American Type Culture Collection USA, NCCS Pune, or from authorized vendors cultured from suitable media supplemented with FBS or FCS or as applicable.

· The cells will be maintained at 37°C under 5% CO2 and 100% humidity in DMEM and supplemented with 10% fetal calf serum and antibiotics (200 μl/ml penicillin G, 200 μg/ml streptomycin, and 2 μg/ml fungizone) and harvested during the logarithmic growth phase.

· After sufficient growth for experimentation, the cells will be trypsinized and plated in 96-cluster well culture plates at a concentration of 1 × 104 cells/well.

· Each well-contained 100μl of cell suspension and the plates will be incubated for 24 h at 37 °C under 5% CO2 to obtain a monolayer culture.

· After 24 h of incubation, the old medium will be removed from each well. Then, a 100-μl eluted volume from the test solution at different concentrations; the positive control; or negative control will be inserted into a 96-cluster well culture plate (8 wells/test material).

· Two 96-cluster well culture plates will be separately prepared to evaluate cell viability using MTT assays and experiments will be repeated in triplicate.

· Following a 24-h incubation period at 37 °C under 5% CO2, the cell viability of both plates will be assessed.

· Assay

· The test materials will be removed from each well of the first plate. Then, 50 μl of MTT reagent (5 mg/ml) will be added and incubated for 2 h at 37 °C in the CO2 incubator.

· The MTT solution will be then discarded, and 100 μl of isopropanol will be added.

· The plates will be placed on a shaker to solubilize the formations of purple crystal formazan.

· The absorbance will be measured using a microplate reader at a wavelength of 570 nm. The results will be used to construct a graph of cell viability percentage against extract concentrations.

3.0 ENDPOINT PARAMETER(S):

· Cell viability


4.0 REFERENCE(S):

4.1 Vajrabhaya, Lo., Korsuwannawong, S. Cytotoxicity evaluation of a Thai herb using tetrazolium (MTT) and sulforhodamine B (SRB) assays. J Anal Sci Technol 9, 15 (2018). https://doi.org/10.1186/s40543-018-0146-0.

4.2 Tolosa L., Donato M.T., Gómez-Lechón M.J. (2015) General Cytotoxicity Assessment by Means of the MTT Assay. In: Vinken M., Rogiers V. (eds) Protocols in In Vitro Hepatocyte Research. Methods in Molecular Biology (Methods and Protocols), vol 1250. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2074-7_26.

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