Determination of non-proliferating cells in culture by combining flow cytometry with stathmokinetics

Colcemid was added to the growth medium of L-cells in monolayer culture. The proliferating cells continued their progression through the cycle up to metaphase, where they were arrested. At different times after Colcemid addition the cells were trypsinized, suspended immediately in a solution of the DNA-specific fluorescent dye Hoechst 33258 and analysed with a flow cytometer. The histograms were evaluated to give the fraction of cells in the 2c peak as a function of time after Colcemid addition. The flux into the 2c compartment being interrupted, the peak content decreased until all proliferating (G1) cells had entered S-phase. With increasing cell density or with increasing time after serum deprivation an increasing fraction of cells remained in the 2c peak at times greater than the normal G1 duration. The possibility of applying this method to the determination of non-proliferating cells in a population is discussed.