Measurement of efflux from G1-phase in a growth factor dependent cell line

Abstract

In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the G1-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.