2.4 | Cell Viability Assay
A549 cells were plated at 5˟103 cells per well in a 96-well plate with 100 µL of the cell suspension for 24 hours at 37°C and 5% CO2 in an incubator. The media was then discarded under aseptic conditions before being replaced with media containing 250 µM of sulindac and varying concentrations of metformin, 400 µM, 800 µM, 1.2 mM, 1.6 mM, and 2.0 mM. Cells were incubated for 48 hours in a 37°C and 5% CO2 incubator. Cell viability was obtained using a CellTiter 96 Aqueous One Cell Proliferation Assay (Promega, Madison, WI) in accordance with the manufacturer’s instructions. This assay used a tetrazolium salt compound that is reduced to a water-soluble formazan dye through dehydrogenase enzymes in metabolically active cells [21, 42]. The formazan dye is quantified by measuring its absorbance at 490 nm with a colorimetric microtiter plate reader (SpectraMax Plus 384, Molecular Devices).
3 | RESULTS
The signal from MitoSOX red in A549 cells demonstrated the production of O2•− by mitochondria under conditions of increased oxidative stress. Figure 1 depicts brightfield and red fluorescent images of A549 cells under the various drug treatment conditions, as well as positive control (AA) and control conditions. The first row is the positive control (AA) group, followed by the control group, and rows three through five are the drug treatment conditions, metformin, sulindac, and combination of metformin and sulindac, respectively. The first column of images contains brightfield images of each treatment group, taken prior to the addition of the fluorescent probe or any drug treatment. The second column of images are taken at 80 minutes, which is 20 minutes after each drug treatment was administered. The third and fourth columns include images taken at 240 minutes and 360 minutes respectively, and visualize increased superoxide anion production as time progresses, over a period of 120 minutes between the two columns. The last column captures intensity after 480 minutes, which is the end point of the experiment. These images demonstrate varying increases in intensities depending on condition. Heightened red fluorescence intensity over time explains increased superoxide production after exposure to treatment conditions.