MTT Cell Proliferation Assay: Testing for Optimal Cell Count

Melvin Onyia
BIOL 4232
Dr. Rachna Sadana

Onyia - LR1

Objective
The primary objective of the experiment was to assess the viability and optimal
proliferation for the cell culture provided. This data will provide the optimal cell
count to use when testing anti-cancer compounds.

Introduction
The assessment of cell viability and proliferation is the foundation for analyzing
cellular activity in regards to cancer treatment. Proliferation assays such as MTT,
XTT, and MTS provide primary screening for compounds that effect normal cell
activity. MTT assay is the primary compound used to test for cell proliferation and
viability. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is
reduce by dehydrogenase enzymes. In result the purple formazan can be quantified.

Materials & Methods

Jurkat, Clone E61 (ATCCTIB152) cells were removed from Coming and Costar
25 cm2 Canted Neck Flask w/Vent Cap under a Laminar flow hood using serological
pipettes & pipette tips and counted under an inverted microscope using a
Hemocytometer to determine concentration. Serial dilution was performed in
triplicates using culture growth medium (RPMI-1640 Medium (ATTCC 30-2001),
10% Fetal Bovine Serum (FBS), 100 U penicillin/0.1 mg/ml streptomycin solution)
into Coming and Costar 96 Well Culture Plate at 100 l per well. Cells were
incubated for 48 hours under normal conditions in a CO 2 incubator at 37 degrees. 10
l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent
was added, and cells were returned to incubator for 3 hours. 100 l of detergent
reagent was added to each well. The plate was wrapped in aluminum foil at room
temperature for 2 hours. The absorbance was read at 570 nM in a microtiter plate
reader.

Onyia - LR1
Results
Data from microtiter plate reader was averaged and standard deviation was
calculated. The experiment was performed twice due to inconsistencies. The data
was graphed plotting the average absorbance vs cells per ml. Figure 1 displays the
data for the first trial and figure 2 displays the graph plotting absorbance vs cell
concentration.

A1
A
B
C
D
E
F
G
H

A2

0.241
0.448
0.338
0.409
0.324
0.307
0.425
0.278

0.239
0.246
0.389
0.283
0.255
0.284
0.32
0.484

A3

Average

STDEV

0.278
0.243
0.306
0.389
0.329
0.331
0.406
0.499

Abs
0.253
0.312
0.344
0.360
0.303
0.307
0.384
0.420

0.022
0.118
0.042
0.068
0.041
0.024
0.056
0.123

0
6211
12422
24844
49688
99376
198751
397502

Figure 1. Raw absorbance data along with average absorbance and

standard deviation.

MTT Cell Proliferation Assay

Absorbance
(570 nm)

0.500
0.400
0.300
0.200
0.100
0.000

Cell Concentration (cells/ml)

Onyia - LR1
Figure 2. Graph plotting the number of cells per ml vs. average
absorbance.

A1
A
B
C
D
E
F
G
H

A2

0.129
0.16
0.251
0.266
0.479
0.383

A3

0.153
0.182
0.193
0.393
0.338
0.435

0.137
0.163
0.178
0.276
0.385
0.376

Cells/ml

Average

St.

0
29219
58438
116875
233750
467500

Abs
0.140
0.168
0.207
0.312
0.401
0.398

Dev.
0.012
0.012
0.039
0.071
0.072
0.032

Figure 3. Raw absorbance data along with average absorbance and

standard deviation.

MTT Cell Proliferation Assay

0.500
0.400
0.300
Absorbance
(570 nm)

0.200
0.100
0.000

Cell Concentration (cells/ml)

Figure 4. Graph plotting the number of cells per ml vs. average

absorbance.

Onyia - LR1
Discussion (8 pts.)
While our first trial provided adequate results there were a number of
inconsistencies in the absorbance readings. The standard deviation was far beyond
reasonable as can be observed in Figure 2. There were also inconsistencies in the
expected slop of the absorbance readings from the data collected. This could be due
to pipetting errors. The second trial displayed more ideal absorbance readings and a
smaller standard deviation. This can be seen in Figure 4. The data shows that the
optimal absorbance is at least 47,000 cells per ml.

Citation & References (3 pts.)

"MTT Cell Proliferation Assay Instruction Guide." ATCC. American Type Culture
Collection, n.d. Web. 10 Feb. 2015.