MTT Cell Proliferation Assay

Onyia - LR1MTT Cell Proliferation Assay: Testing for Optimal Cell CountMelvin OnyiaBIOL 4232Dr. Rachna Sadana

ObjectiveThe 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.

IntroductionThe 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,CloneE61(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 CO2 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.

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. A1A2A3Average AbsSTDEV

A0.2410.2390.27800.2530.022

B0.4480.2460.24362110.3120.118

C0.3380.3890.306124220.3440.042

D0.4090.2830.389248440.3600.068

E0.3240.2550.329496880.3030.041

F0.3070.2840.331993760.3070.024

G0.4250.320.4061987510.3840.056

H0.2780.4840.4993975020.4200.123

Figure 1. Raw absorbance data along with average absorbance and standard deviation.

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

A1A2A3Cells/mlAverage AbsSt. Dev.

A------

B------

C0.1290.1530.13700.1400.012

D0.160.1820.163292190.1680.012

E0.2510.1930.178584380.2070.039

F0.2660.3930.2761168750.3120.071

G0.4790.3380.3852337500.4010.072

H0.3830.4350.3764675000.3980.032

Figure 3. Raw absorbance data along with average absorbance and standard deviation.

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

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.

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