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.