A new in vitro method using fura-2 for the quantification of endothelial cell injury

Endothelial cell injury was determined by a new method based on measuring a specific release of fura-2, a fluorescent calcium indicator. We examined the cytotoxicity of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) on cultured endothelial cells using the fura-2 assay. The new assay was compared to traditional assays for cytolysis such as measuring the release of 51Chromium (51Cr) or lactate dehydrogenase (LDH). The fura-2 method were able to detect minor and early phase membrane damage in endothelial cells due to the hydroperoxide. Significant release of fura-2 was detected by 5, 10, 25 and 50 mu/ml of 15-HPETE (5, 10 and 25 micrograms/ml p less than 0.01; 50 micrograms/ml, p less than 0.001) as early as 1 hour after the start of the 15-HPETE treatment. The fura-2 assay was so sensitive to the endothelial cell injury that the damage of the cells due to 15-HPETE at the dose of as low as 5 mu/ml could be detected in as early as 30 min. On the other hand, in the traditional 51Cr or LDH release assay, only the cell damage caused by high dose (50 mu/ml) of 15-HPETE could be detected in as late as 6 hours after the start of 15-HPETE exposure. The new method may be useful for the approach to searching injurious substances or their antagonists to several kinds of cells.