Cell Injury Markers

Caspase-3 activity (NucView 488 Caspase-3 Assay Kit; Biotium, Hayward, CA) and PI uptake (Sigma-Aldrich) were measured in HEK cells in a 6-well tissue culture plate. HEK cells were transfected with 2.0 μg mouse lipase in pcDNA 3.1, as discussed above. GTL (600 μmol/L) was then added for 1.5, 3.0, or 5.0 hours, as discussed above. At the end of treatment, cells were stained with 4 μmol/L NucView 488 caspase substrate, 2 μL/mL PI, and 1 μg/mL Hoechst 33342 (ImmunoChemistry Technologies, LLC, Bloomington, MN) and incubated in the dark for 15 minutes at room temperature. Cell pellet was disrupted in phosphate-buffered saline by sonication, and bright green (NucView 488 Caspase-3 substrate; excitation/emission, 485/515 nm), red (PI; excitation/emission, 535/617 nm), and blue (Hoechst 33342; excitation/emission, 345/460 nm) fluorescence values were measured using the VersaFluor Fluorometer (Bio-Rad, Hercules, CA). Results were expressed as fluorescence unit for caspase-3 activity or PI uptake and normalized, with the fluorescence of Hoechst 33342 as control. Measurement of caspase-3 activity and PI uptake in HEK cells (without overexpression of mouse lipase) after 100 μmol/L LA treatment was done, similar to GTL exposure, as detailed above.

Cell death was quantified using a colorimetric cytotoxicity assay for LDH release in the media. Briefly, absorbance at 490 nm and background absorbance at 620 nm were measured in the media using the LDH assay kit (Roche Applied Sciences, Indianapolis, IN) after 4 hours of treatment. Results were expressed as a percentage of total LDH leakage, normalized to that measured in cells lysed with 1% Triton X-100. PI uptake by cells, as a measure of dead necrotic cells, was quantified as previously described^{1, 55} using a flourimetric method.