4.2.2 (a) Detection of Cell Viability Using MTT and Cell Titer blue
For dose dependent viability assessment, cells were stimulated with different concentration (5µg, 10µg, 15µg) of purified Rv2615c protein for 24h. For checking the viability in time dependent manner, unstimulated cells and cells stimulated with (10µg/ml) Rv2615c/(40ng/ml) LPS/(2μM) Staurosporine were incubated for 16h, 24h and 48h. Staurosporine is an alkaloid derivative from the bacterium Streptomyces staurosporeus which has been extensively been used as an inducer of apoptosis because it inhibits many different kinases.
Cells were exposed to either CellTiter Blue Reagent (20 mL/well) or MTT (5 mg/ml) and incubated for 4 hours at 37 °C and 5% CO2. Tetrazolium rings can be transformed into purple, insoluble formazan crystals by active dehydrogenases found in live cells. The crystals were dissolved in 100 µL of DMSO and the overall activity of bio-reductase enzyme was measured spectrophotometrically at (570-690) nm. The CellTiter-Blue Reagent constitutes of resazurin in a buffer solution. Resorufin (dark pink), which is extremely fluorescent and whose absorbance may be measured spectrophotometrically at (570–600) nm, can be reduced from resazurin (dark blue) by viable cells. The percentage of cell viability was calculated using the acquired absorbance values. Cells cultured in medium alone were thought to be completely viable.