DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidiumbromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.
Key terms: Ethidium bromide, Hoechst
33258, G1 cell fluorescence, basis nuclear proteinsWe have reported on the application of flow cytometry (FCM) to the analysis of the DNA content of Euglena cells: fluorescence intensity of Euglena stained with ethidium bromide (EB) increased during the lag phase preceding exponential growth, and decreased during the stationary stage when G1 cells returned to a quiescent state (5).The decreased EB binding observed in cells in transition from exponential to stationary phase could be due either to a loss of DNA per cell or to a decrease in the accessibility of the DNA to EB binding and to intercalating fluorochromes in general. To distinguish between these possibilities, DNA staining by the intercalating dyes acridine orange (AO), DAPI, and EB (9,14,24) and by the externally binding dye Hoechst 33258 (9) ha: been compared.
MATERIAJS AND METHODS Experiments were performed on Euglena gracilisKlebs strain Z. Cells were grown at 23°C in the light in a synthetic medium containing minerals, lactic acid, and vitamins B1 and B12 (4).Colorimetric DNA measurements were performed with the diphenylamine method (3,17). For FCM DNA measurements, cells were fixed and chlorophyll extracted in absolute ethanol. Before staining, cells were first washed with glass-distilled water.Ethidium Bromide Staining Fixed calf thymocytes (Ortho Diagnostic Systems, NJ) were added to samples as a n internal standard. Cells were first incubated in RNase (Boehringer Mannheim, Ingelheim amj Rhein, FRG) 1 mgiml in a phosphate buffer 0.2 M, pH 7, for 1 hr, either at 37°C or at 50°C. RNase was either boiled or not, and either added or not with Triton XlOO (0.1%). Cells were twice washed with glass-distilled water and stained with EB (Calbiochem, LaJolla, CA) 50 pglml or 10 pg/ml. For the lowest fluorochrome concentration, the effect of Triton XlOO (0.1%), NaCl (0.15 M), MgCl2 (2 mM), and HC1 (final concentration ranging between 0.02 N to 0.2 N) were tested. Cells were analyzed within 1 hr.
Acridine Orange StainingCells (lo6 per sample) were first incubated in RNase for 1 hr at 37°C. ...
