MTT ASSAY FOR CELL CYTOTOXICITY

_________________________________________________
PRINCIPLE

MTT (3-4, 5 dimethylthiazol-2yl-2, 5-diphenyl tetrazolium bromide) assay, is based on the ability
of a mitochondrial dehydrogenase enzyme of viable cells to cleave the tertrazolium rings of the pale
yellow MTT and form a dark blue colored formazan crystals which is largely impermeable to cell
membranes, thus resulting in its accumulation within healthy cells. Solubilization of cells by the addition
of detergents (DMSO) results in the liberation of crystals which are solubilized. The number of surviving
cells is directly proportional to the level of formazan product created. The color can be quantified using a
multi-well plate reader.

MATERIALS REQUIRED

DMEM medium, Fetal Bovine Serum (FBS) and antibiotic solution were from Gibco (USA),
DMSO (Dimethyl sulfoxide) and MTT (3-4,5 dimethylthiazol-2yl-2,5-diphenyl tetrazolium bromide) (5
mg/ml) were from Sigma, (USA), 1X PBS was from Himedia, (India). 96 well tissue culture plate and
wash beaker were from Tarson (India).

PROCEDURE

Cell culture

HSC-3 (human oral carcinnoma cells) cell line were cultured in liquid medium (DMEM)
supplemented 10% Fetal Bovine Serum (FBS), 100 u/ml penicillin and 100 µg/ml streptomycin,
and maintained under an atmosphere of 5% CO2 at 37oC.

MTT Assay

The S1 sample was tested for in vitro cytotoxicity, using HSC-3 (human oral carcinnoma
cells) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Briefly, the cultured HSC-3 cells were harvested by trypsinization, pooled in a 15 ml tube. Then,
the cells were plated at a density of 1×105 cells/ml cells/well (200 µL) into 96-well tissue culture
plate in DMEM medium containing 10 % FBS and 1% antibiotic solution for 24-48 hour at
37°C. The wells were washed with sterile PBS and treated with various concentrations of the S1
sample in a serum free DMEM medium. Each sample was replicated three times and the cells
were incubated at 37°C in a humidified 5% CO2 incubator for 24 h. After the incubation period,
MTT (20 µL of 5 mg/ml) was added into each well and the cells incubated for another 2-4 h until
purple precipitates were clearly visible under an inverted microscope. Finally, the medium
together with MTT (220 µL) were aspirated off the wells and washed with 1X PBS (200 µl).
Furthermore, to dissolve formazan crystals, DMSO (100 µL) was added and the plate was
shaken for 5 min. The absorbance for each well was measured at 570 nm using a micro plate
reader (Thermo Fisher Scientific, USA) and the percentAge cell viability and IC50 value was
calculated using GraphPad Prism 6.0 software (USA).

RESULT

A. OD Value at 570 nm
Control Mean OD value: 0.584

S. Tested sample OD Value at 570 nm

No concentration (μg/ml) (in triplicates)
1. Control 0.561 0.586 0.606
2. 100 μg/ml 0.092 0.070 0.149
3. 90 μg/ml 0.219 0.237 0.239
4. 80 μg/ml 0.237 0.228 0.244
5. 70 μg/ml 0.261 0.260 0.251
6. 60 μg/ml 0.286 0.299 0.269
7. 50 μg/ml 0.301 0.308 0.359
8. 40 μg/ml 0.360 0.377 0.368
9. 30 μg/ml 0.359 0.403 0.412
10. 20 μg/ml 0.465 0.463 0.451
11. 10 μg/ml 0.560 0.500 0.520
 lo g - d o s e v s r e s p o n s e
0 .8
C o n tro l

S a m p le S 1  g /m l
0 .6
O D at 570 nm

0 .4

0 .2

0 .0
0 50 100 150

S a m p le S 1  g /m l

B. Cell Viability (%)

S. No Tested sample Cell viability (%) Mean

concentration (in triplicates) Value (%)
(μg/ml)
1. Control 100 100 100 100
2. 100 μg/ml 15.75 11.98 25.51 17.74
3. 90 μg/ml 37.5 40.58 40.92 39.66
4. 80 μg/ml 40.58 39.04 41.78 40.46
5. 70 μg/ml 44.69 44.52 42.97 44.06
6. 60 μg/ml 48.97 51.19 46.06 48.74
7. 50 μg/ml 51.54 52.73 61.47 55.24
8. 40 μg/ml 61.64 64.55 63.01 63.06
9. 30 μg/ml 61.47 69.00 70.54 67.00
10. 20 μg/ml 79.62 79.28 77.22 78.70
11. 10 μg/ml 95.89 85.61 89.04 90.18
 150

C e ll v ia b ilit y ( % )
100

50

0
l l l l l l l l l l l
o /m /m /m /m /m /m /m /m /m /m
tr g g g g g g g g g g
n          
o 0 0 0 0 0 0 0 0 0 0
C 0 9 8 7 6 5 4 3 2 1
1

S a m p le S 1  g /m l

C. IC50 Value of tested sample: 49.73 μg/ml

log(inhibitor) vs. normalized response -- Variable slope

log(inhibitor) vs. normalized response -- Variable slope
Best-fit values
LogIC50 1.697
HillSlope -2.058
IC50 49.73
Std. Error
LogIC50 0.01782
HillSlope 0.2012
95% Confidence Intervals
1.660 to
LogIC50 1.733
-2.470
to -
HillSlope 1.646
45.72 to
IC50 54.09
Goodness of Fit
Degrees of Freedom 28
R square 0.9101
Absolute Sum of Squares 2106
Sy.x 8.673
A. Formation of formazan crystals in control cells and S1 sample treated cells

Before MTT treatment After MTT treatment

Control cells

S2 sample100 μg/ml