Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

Abstract

A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

“…3c). Similar transients have been reported previously for Nicotiana tabacum (Nicoloso et al, 1994) and CHO (Leelavatcharamas et al, 1996) cells, illustrating the synchronizing effect that exposure to new medium can have on asynchronous cultures. Synchrony was maintained over about three cell cycles in the N. tabacum cultures; however, possibly because of the relatively high proportion (ca.…”

“…Chopping also avoids the delay of several hours for enzyme digestion between sampling and final recovery of nuclei, thus reducing the risk of changes in the cell cycle distributions. Although mechanical maceration has been used in previous flow cytometry studies to isolate nuclei from intact suspended plant cells (Galbraith, 1990;Nicoloso et al, 1994), considerable difficulty was encountered in this work with cell debris produced during the chopping process. These problems were exacerbated by the small size of the S. aviculare nuclei.…”

“…Methods have been developed in recent years based on plant protoplast, nuclear and pollen suspensions (Brown and Bergounioux, 1989;Galbraith et al, 1983Galbraith et al, , 1984Harkins and Galbraith, 1987). Current applications of flow cytometry to plant cells include DNA and RNA analysis, detection of transgene expression, karyotyping, cell counting, studies of chloroplasts, cell membranes and cell wall regeneration, evaluation of mitochondrial activity, measurement of secondary metabolite accumulation, and selection of particular cells or sub-cellular organelles of interest (Adamse, 1990;Bergounioux et al, 1992;Conia and Muller, 1989;Galbraith, 1989;Galbraith et al, 1995;Nicoloso et al, 1994;Petit, 1992;Sakamoto et al, 1994;Schröder and Petit, 1992). Methods for BrdU labeling of plant cells were first described by Levi et al (1987) to determine the proportion of S-phase cells in pea root meristems using microscope photometry.…”

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

“…To determine the nuclear DNA content we used flow cytometry, which offers a rapid method for the analysis of the DNA content in thousands of plant nuclei (Dole2al 1991, Nicoloso et al 1994, Valkonen 1994. The flow cytometry (FCM) analysis of nuclear DNA content consists in measuring the fluorescence associated with each nucleus after stoichiometric staining with DNA specific fluorochrome and comparing the results with a reference DNA standard.…”

Are polyamines directly involved in silymarin production in the milk thistle [Silybum marianum (L.) Gaernt]?

“…Cell nuclei were released by hypotonic lyses of isolated protoplasts (Cacho 1991) using previously fixed cells (Nicoloso et al 1994). The protoplasts were macerated for 30 min in ice-cold LB01 lysis buffer (Dolezel et al 1989), filtered through a nylon mesh (pore diameter 42 lm) and centrifuged (3,000 rpm, 5 min).…”