Hb. Oral et al., A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury, ENDOTHELIU, 6(2), 1998, pp. 143-151
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.