A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury

A rapid and sensitive quantitative fluorometric assay was developed to meas ure the response of endothelial cells to hydrogen peroxide (H2O2). The resp onse of an endothelial cell-derived cell line, EA-hy-926, or human umbilica l vein endothelial cells to H2O2 was determined using calcein-AM, a dye whi ch becomes fluorescent upon cleavage by intracellular esterase(s). The abil ity of the cells to take up and convert calcein-AM was measured directly in the wells of 96-well flat-bottomed tissue culture plates with cell monolay ers using a computerised microplate fluorimeter, or in cell suspensions usi ng flow cytometry. The results obtained by these techniques were compared w ith each other and with a standard Cr-51 release cytotoxicity assay. We fou nd that calcein-AM is a highly sensitive probe for measuring H2O2-mediated cell injury, as it can not only detect the irreversible cyotoxicity measure d by Cr-51 release assay, but can also distinguish sublethal and reversible injury seen at low H2O2 concentrations.