A Cellular Viability Assay to Monitor Drug Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 292

mitochondrial enzymes; however, later studies showed that MTT
reduction also takes place in microsomal and cytosolic fractions
by oxido-reductase type enzymes transferring two electrons to
MTT, most likely through reduced forms of NADH and NAPDH
( 7-9 ). The reduced form of MTT is an insoluble dark colored
product, called formazan which upon re-solubilization can be
quantified by measuring the absorbance at wavelengths in the
range 550-600 nm. Before using the MTT assay for evaluation of
a putative toxicity of a drug it is recommendable to perform a pilot
study to test MTT reduction rates as a function of the cell seeding
density. This will give an idea of the number of cells required to
get a significant and detectable signal of a magnitude that later
allows for detection of a decreased MTT reduction. The use of too
many cells per well may lead to the depletion of nutrients such as
glucose that has been shown to affect cellular MTT reduction
rates ( 9 ). The magnitude of the MTT signal is often approaching
a linear correlation with cell density ( 6 ), which is also evident from
the data presented here using lymphoblastoid cells (Fig. 1 ).
Putative toxic effects are investigated by exposing a set of cells
to celastrol in a period prior to addition of MTT and then taking

0.3

0.25

0.2

0.15

0.1

0.05

0
0.0E+00 5.0E+03 1.0E+04 1.5E+04 2.0E+04 2.5E+04 3.0E+04 3.5E+04 4.0E+04

Number of cells per well

Fig. 1. Correlation between MTT reduction and cell seeding density. Lymphoblastoide cells were seeded at densities from
4.0E+04 cells per well to 6.2E+02 cells per well in 90 µl culture media in a 96-well microplate. Following overnight
incubation, 10 µl MTT solution was added (final concentration 0.5 µg/µl). The MTT reduction was stopped after 4 h by
addition of 100 µl solubilization buffer. Solubilized MTT was quantified by measuring the absorbance at 595 nm with
reference at 655 nm ([A 595 -A 655 ] and plotted as a function of cell density). Each value represents the mean of three experi-
ments and the mean coefficient of variance for all cell densities was 16%.