MTT Assay for Cell Viability

The cells were plated in 24 well culture plates (25000 cells/500 µl/well for all the cell lines except Raw264.7, which was seeded with 40,000 cells/well density) and incubated in CO₂ incubator. Next day, treatment was given according to the experimental requirement. Further, at respective time points, 50 µl MTT solutions from the Stock (5 mg/ml) was added and cells were incubated in CO₂ incubator in the dark for 2 hrs. The medium was removed and Formazan crystals formed by the cells were dissolved using 500 µl of DMSO followed by transfer in 96 well plate. The absorbance was read at 570 nm using 630 nm as reference wavelength on a Multiwell plate reader (Biotech Instruments, USA). Reduced formazan quantification was done with Formazan standard. At each respective time points cell numbers were counted with a Neubauer-improved counting chamber (Paul Marienfeld GmbH & Co. KG, Germany) under 10X objective, and 10X eye piece magnification with compound light microscope (Olympus CH30, Japan). After MTT incubation Formazan images were captured under bright field (20X objective) with inverted microscope (Olympus, Japan). All the parameters were recorded under similar experimental conditions.