Synchronization of mammalian cells by selection and additional chemical block studied by DNA distribution analysis and BrdUrd‐Hoechst 33258‐technique

Abstract

Ehrlich ascites tumour cells growing in vitro in suspension culture were separated according to volume by the technique of velocity sedimentation in a zonal rotor with a reorienting gradient. Using DNA distribution analysis the sedimentation pattern of the cells could be analysed in detail. With appropriate conditions it was possible to separate pure G, cells. Samples could also be obtained which were enriched in S or G, + M cells. The main limitation of the selection in this type of rotor

Flow cytometric analysis of G1‐ and G2/M‐phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements

“…In fact, from the data in Figures l a and 2 (exponentially growing cells), the fraction of cells in M-phase was estimated to be NM/N = 0.024 t 0.003, whereas the fraction of cells in G,A was estimated to be NG,A/N = 0.046 k 0.003 (NG1A1 = 0.013 and NG,A/N = 0.033). With the exponential age distribution the duration of GIA,-phase was calculated for comparison (9). The calculated durations of the G,A,-and G,A,-phases were t c , A , = 10 min and tGIA, = 15 min, a very good agreement with the data obtained by the analysis of colcemid released cells shown on Figure 5.…”

The importance of G1/S-border and mitosis in the fixation of potentially lethal damage

“…After incubation, aphidicolin was washed out and cells were resuspended in fresh medium. Three hours after block release the cells were in mid S-phase and after 6 h mostly in G2 + M-phase (Ntisse 1982). The durations of the cell cycle phases were: TG1 = 2.4 h, TS = 5.0 h, and TG2 + M = 2.6 h. Cell cycle distributions were checked by measuring DNA distributions of cells in a flow cytometer (ICP 21, Phywe, Germany) after staining with the fluorochrome Hoechst 33258 (Riedel-de Haen, Germany) according to B6hmer (B6hmer 1979).…”