Mtt Assay Lab Report

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MTT assay was used to analyze cell viability of MCF 10A, MCF 7 and MDB-MB 231 cell lines in control and 10mM inorganic phosphate treated groups. MTT assay is a colorimetric assay for evaluating cell metabolic activity. MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) is a yellow aqueous tetrazolium dye. In living cells with active mitochondria, the mitochondrial enzymes such as succinate dehydrogenase (NADPH dependent oxidoreductase enzyme) reduce this dye to formazan, a purple-colored water-insoluble complex (Gomez et al., 1997). The intensity of absorption of the purple complex is read typically at 595nm using a spectrophotometer and is a direct measure of mitochondrial activity.
The materials and equipment required to perform this assay included MTT at 5mg/ml in calcium-free PBS (Cat #M 2128-1gm, Lot #MKBS4732V, Sigma Aldrich), stored at 40C in the dark, isopropanol (Cas # 67-63-0, J.T Baker, microcentrifuge tubes- 1ml (Catalog #211511, Lot #447425-Y23093, Avant, LPS, Rochester, NY), a 96-well plate for reading (Ref #353072, Falcon, Corning-Life Sciences, Durham, NC), pipette tips/repipettor (1ml, 200ul,Ref #702390, Eppendorf, Germany), centrifuge (Centrifuge 5415D, Eppendorf, Germany) and spectrophotometer (EL808 Microplate, BioTek Winooski, VT).
Samples were …show more content…

Silver nitrate solution (5%) was added (1ml/well) and cells incubated for 60min under a UV light source. At the end of 60 min exposure, the wells were rinsed with several changes of 1ml/well distilled deionized water. After this, 5% sodium thiosulfate was added (1ml/well) and cells were incubated at room temperature for 5min. This was followed by several rinses of distilled deionized water and the wells were photographed under phase contrast to see visible black/brown

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