PHARMACOLOGICAL SCREENING:
MTT ASSAY
CHAPTER-3.2.2
3.2.2.1
INTRODUCTION
The measure of toxicity to the living cells is referred to as Cytotoxicity. It is the killing
capacity of the agents. Cytotoxicity assays are widely used in in vitro toxicology studies
for screening of drugs and determination of cytotoxicity of chemicals.
3.2.2.2
ASSAY PROCEDURES
Numerous assays procedures (Celis, 2006) used to determine the effect of drug on cells
propagated in vitro are enumerated below:
a)
Dye exclusion test using trypan blue: It is a simple and inexpensive test, which
is based on the difference in permeability of living and dead cells to trypan blue dye. In
the dead cells there is an uptake of the dye and they appear dark blue coloured, but the
viable cells remain unstained and appear clear. This helps in determining the cell
membrane integrity.
b)
3[H]-Thymidine
incorporation assay: Measuring the uptake of tritium-labelled
thymidine into replicating DNA measures the toxicity of agents. Since thymidine is taken
up by actively dividing cells, but dead cells are devoid of this propensity.
c)
ATP cell viability assay: ATP can be quantitated in a luminometer by measuring
the light generated using luciferase-luciferin reagent. Dying or dead cells exhibit
significantly lower levels of ATP when compared to viable cells, due to loss of cell
integrity.
d)
Alamar Blue assay: In this method, the reducing environment of proliferating
cells is monitored. The procedure is based on the potential of mitochondrial reductase to
convert nonfluorescent resazurin to fluorescent resorufin.
e)
Sulforhodamine assay: The method is based on binding of the dye
(sulforhodamine
B) to basic amino acids of cellular proteins. The colorimetric evaluation
Ph.D. Thesis (Akranth Marella)
Page 251
PHARMACOLOGICAL SCREENING:
MTT ASSAY
CHAPTER-3.2.2
provides an estimate of total protein mass, which bears a direct relation to the cell
number.
f)
MTS assay: The assay is based on the principle that yellow coloured tetrazolium
salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) in presence of
electron coupling reagent phenazine methosulfate is reduced to purple colour formazan
by living cells. The tetrazolium ring is cleaved in active mitochondria by reductase
enzymes; hence, the process is specific to living cells only. Quantification of the colour
of this solution at a specific wavelength, measured using a UV spectrophotometer, is the
measure of cytotoxicity of the compound (Mosmann, 1983).
N
NH
N
N
N
N
Mitochondrial
reductase
N
N
N
N
S
S
Br
Yellow coloured salt
3.2.2.3
Purple coloured formazan
METHODOLOGY
Cytotoxicity of the present set of compounds was determined by MTT assay using Vero
cell lines. The tests were performed on compounds having IC50 less than 5 µM and their
selectivity index was determined. 50% cytotoxic concentration (CC50) values represent
the concentration of compound required to kill 50% of the fibroblast cells. The selectivity
index (SI) was determined using the formula
SI = CC50/IC50
Ph.D. Thesis (Akranth Marella)
Page 252
CHAPTER-3.2.2
PHARMACOLOGICAL SCREENING:
MTT ASSAY
MTT- (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide was dissolved in
PBS at 5 mg/mL and filtered to sterilize. MTT stock solution (10 µL/100 µL medium)
was added to all wells and plates were incubated at 37°C for 4 h. Acid-isopropanol
(100 µL of 0.04 N HCl in isopropanol) was added to all wells and mixed thoroughly to
dissolve the dark blue crystals. After a few minutes at room temperature (to ensure that
all crystals were dissolved), the plates were read on UV spectrophotometer, using a test
wavelength of 570 nm, and reference wavelength of 630 nm. Plates were read within 1 h
of adding isopropanol.
The compounds having IC50 less than 5 µM were tested for their selectivity index (SI).
Since, none of the compounds in SBP series had IC50 less than 5 µM so SI was
determined only for CPP and TPP series, which are presented in Table 4 and 5
respectively.
Ph.D. Thesis (Akranth Marella)
Page 253