4.4. 2D Cell Viability Assay (MTT)

The MTT assay is the standard assay used to measure cytotoxicity in adherent cells in drug discovery [47]. This assay has been used extensively to report on the activity of many marine natural compounds discovered at HBOI. This assay is performed by seeding the cells and allowing them to adhere, then on the next day treating with compound for 72 h and assessing the viability of cells at the end of the experiment. This assay relies on the metabolization of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its insoluble formazan, resulting in the formation of purple crystals. When cells lose their viability, they lose their ability to metabolize the compound, resulting in a marked change in color that is easy to measure using absorbance in a plate reader [47]. Thus, this standard assay and treatment time were used to test the compound for 2D cytotoxicity.

Cells were plated on a clear flat bottomed 384-well tissue culture plate at a concentration of 3000 cells per well in a volume of 30 μL/well and allowed to adhere overnight. At the end of this incubation, 30 μL of medium containing treatment at two times the final concentration was added. Enriched fraction library samples and pure compounds with low cytotoxicity (micromolar) were tested at 5 μg/mL. Pure compounds with known cytotoxicity at the nanomolar range were tested at 0.5 μg/mL. Other samples tested included 100 ng/mL super killer TRAIL and the known inducer of apoptosis microsclerodermin A at a concentration of 2.4 μg/mL. Plate controls included 10 μM ABT737, 5 μg/mL 5-fluorouracil, 0.5 μg/mL doxorubicin, media alone and solvent controls. The same compounds were tested in the 2D assay at the same concentrations used in the 3D assay. The cells were then incubated for 72 h at 37 °C and 5% CO₂. After this incubation, 125 μg MTT was added to each well. The cells were then incubated for 3 h at 37 °C followed by centrifugation. The supernatant was removed and 100 μL acidified isopropyl alcohol (1:500 solution of hydrochloric acid to isopropanol) was added to each well to dissolve the crystals. The absorbencies of these solutions were measured at 570 nm with a plate reader (NOVOstar, BMG Labtech Inc., Durham, NC, USA). The resulting absorbencies were normalized against ethanol-treated cells using Microsoft Excel (Redmond, WA, USA).