Flow cytometry and cell proliferation kinetics

Flow cytometric techniques are presented which allow to determine parameters of cell proliferation kinetics by means of histogram sequences after special manipulations of the cell culture under investigation: (a) In the stathmokinetic method metaphase blocking agents are applied which allow the cells of the population to continue progression through interphase and accumulate at 4C DNA content. The development of DNA specific histograms during this process is analysed as to the G1 phase duration and the fraction of nonproliferating cells. (b) In the BUdR/Hoechst method the suppression of Hoechst fluorescence after BUdR incorporation during S phase is taken as a means for inducing a temporal change of histogram shapes without perturbing the cell cycle progression of the population. This temporal development of histogram shapes is analysed as to phase duration, whole cycle time and fraction of nonproliferating cells. (c) By combining the BUdR/Hoechst technique with a simultanous DNA specific stain and analysing with a two-parametrical flow cytometer, more information is obtained from each histogram after BUdR incorporation: The location of cells in the cycle at the beginning of the experiment, the cycle stage at cell harvest, and from this the distance and velocity of progression through the cycle during drug incubation. By introduction of these dynamic methods flow cytometry has become a powerful tool for the study of cell proliferation kinetics in culture.