Flow cytometric detection of a two-step cell death induced by hyperthermia

A human leukaemic cell line (REH) was subjected to various temperatures approximately greater than 42 degrees C for various time intervals; the cells were stained with a mixture of ethidium bromide and acridine orange, and red and green fluorescence were analysed by flow cytometry. Nontreated cells appeared as one cluster (V) in the biparameter histograms, but with time of heat treatment, two further discrete clusters (D1,D2) of cells appeared successively. They were distinguished by both the degree of red and green fluorescence. The kinetics of transit from one cluster to the other was dependent on temperature, the time lag between both steps becoming shorter with higher temperatures. It was shown previously that the same effect occurred during incubation with various cytostatic agents, and that only the D2 stage correlated with the stage of cell death monitored by the usual trypan blue exclusion test. Therefore the ethidium bromide technique seems to monitor an earlier stage of cell death. The decrease in the number of dye-excluding (V) cells during heat exposure occurred in two phases. After an initial decrease a plateau of number of dye-excluding cells was reached; the duration and level of this plateau depended on the temperature. The plateau was followed by a second phase where the remaining cells ceased to exclude the dye.