Introduction

For assaying a compound toxicity (e.g anticancer compound) in in-vitro cell culture experiment, generally we used MTT assay it is commonly know as cell viability,and proliferation assay MTT assay is the most common reaction in the cell culture procedure. MTT stands for (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide).

Mechanism

The assay is depend on the cleavage of the yellow tetrazolium salt MTT in to the purple formazan crystals by metabolic active cells. This is also known as colorimetric assay. This cellular decline involves the pyridine nucleotide cofactors NADH and NADPH . The formazan crystals formed are solubilized and the consequential colored elucidation is quantified using a scanning multiwell spectrophotometer (ELISA reader). This ensures a high degree of precision, enables online computer processing of the data: Data collection,Calculation and Report generation and, thus, allows the rapid and well-situated handling of a high number of samples.

Materials:
PBS-Phosphate buffer saline
MTT (5 mg/ml in PBS) - filter and keep dark, prepare freshly Acidic isopropanol (0.1N HCl in absolute isopropanol) 96-well plate (flat bottom).

Procedure:

1. Plate cells (104 - 106 cells) in 200 ml PBS in 96-well (flat bottom).
2. Add 20 ml of MTT solution, mix well.
3. Incubate for 4 hour in 37ºC in dark.
4. Remove aliquot for analysis; add 200 ml acidic isopropanol and mix well.
5. Incubate additional 1h in 37°C in dark.
6. Read plate in ELISA Reader - measure OD in 570nm (background wavelength is
630nm).

Application

Cell proliferation and viability assays are of meticulous significance for routine applications.
It is a reliable ,sensitive, quantitative and automated methods led to the development of standard assays.

About Author / Additional Info:
Hello,
I am goldy yadav m.tech biotechnology, RGPV,Bhopal