Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.     

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.     

Cell cycle operation during batch growth of fission yeast populations

Batch cultivation provides a continuous sequence of different environments useful for studying responses of cell cycle controls. Flow cytometry measurements have been made of the frequency functions for protein, RNA, and DNA at different times during batch growth of the fission yeast Schizosaccharomyces pombe. The mean cellular protein and RNA contents and their variances tend to increase with increasing population specific growth rates. Analysis of the mid-exponential phase DNA frequency function data indicates that DNA synthesis occupies 12% of the total cell cycle time and is completed at the same time as cell separation. Coordination of DNA synthesis and cell separation is less precise when population growth rate is low in late lag and early stationary phases.     

A Rapid Automated Stathmokinetic Method For Determination of In Vitro Cell Cycle Transit Times

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G₁. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G₂+ M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.     

Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide     

Population analysis of a commercial Saccharomyces cerevisiae wine yeast in a batch culture by electric particle analysis, light diffraction and flow cytometry

Data from electric particle analysis, light diffraction and flow cytometry analysis provide information on changes in cell morphology. Here, we report analyses of Saccharomyces cerevisiae populations growing in a batch culture using these techniques. The size distributions were determined by electric particle analysis and by light diffraction in order to compare their outcomes. Flow cytometry parameters forward (related to cell size) and side (related to cell granularity) scatter were also determined to complement this information. These distributions of yeast properties were analysed statistically and by a complexity index. The cell size of Saccharomyces at the lag phase was smaller than that at the beginning of the exponential phase, whereas during the stationary phase, the cell size converged with the values observed during the lag phase. These experimental techniques, when used together, allow us to distinguish among and characterize the cell size, cell granularity and the structure of the yeast population through the three growth phases. Flow cytometry patterns are better than light diffraction and electric particle analysis in showing the existence of subpopulations during the different phases, especially during the stationary phase. The use of a complexity index in this context helped to differentiate these phases and confirmed the yeast cell heterogeneity.     

Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Flow Cytometric Analysis of Electronic Nuclear Volume and DNA Content in Normal Mouse Tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE QuantaTm for measuring EV and DNA content of different normal mice tissues.Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Genome Size, Complexity, and Ploidy of the Pathogenic Fungus Histoplasma capsulatum

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus Histoplasma capsulatum was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, α-tubulin, and β-tubulin). G186AS was found to have a genome of approximately 2.3 × 10⁷ bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 × 10⁷ bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1n genome estimate to yield a DNA index (DNA per cell/1n genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with α- and β-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.     

Flow cytometry of Escherichia coli for process monitoring

A laser flow cytometer was used to study different Escherichia coli populations under various cultivation conditions. A host strain E. coli 5K was analyzed for cell size, protein and DNA-content during continuous cultivation. Also, a recombinant E. coli 5K(pHM12) strain (used for the intracellular production of penicillin-G acylase) was studied in regard to gene expression using different cytometric techniques. An argon ion laser (30 mW) and a 100 W high-pressure mercury lamp were used as light source in the cytometer. A new fluorogenic staining technique for intracellular penicillin-G acylase is described.Recombinant E. coli temperature sensitive cells were analyzed for intracellular fusion protein production due to temperature induction.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Use of flow cytometry in the measurement of cell mitotic cycle

Variations in many cellular characteristics during the cell cycle can be analyzed simply and directly by flow cytometry. Using multiparameter analysis of DNA content, RNA content, cell size and 5-bromodeoxyuridine (BrdUrd) incorporation, it is now possible to define cells' positions in the cell cycle with a precision previously unimaginable. It is also possible, by using the sorting function of the flow cytometer, to separate populations in different phases of the cell cycle for biological and biochemical studies. This review describes the technical aspects of flow cytometric instrumentation, DNA staining procedures, and the cytometric applications of both in cell cycle analysis including some of the more innovative, new approaches with antibody against BrdUrd.     

New at-line flow cytometric protocols for determining carotenoid content and cell viability during Rhodosporidium toruloides NCYC 921 batch growth

Rhodosporidium toruloides NCYC 921 batch growth was monitored as a means to evaluate the yeast biomass potential as a source for the production of carotenoids and other lipids.Carotenoid content, cell viability and size were assessed by multiparameter flow cytometry. The saponifiable lipid fraction was assayed by gas–liquid chromatography.The carotenoid production increased during the stationary phase, reaching 78 μg/g while the total fatty acid content attained 32% (w/w) at the end of the fermentation. The fatty acid profile was suitable for biodiesel purposes.As the yeast cells entered the stationary phase, the proportion of cells with depolarised mitochondrial membrane and cells with permeabilised cytoplasmic membrane increased, attaining 65% and 14%, respectively. Nevertheless, a high proportion of cells (82%) showed esterase activity.These results demonstrated that flow cytometry can be a powerful at-line technique to monitor the total carotenoids and cell viability during the yeast growth, being useful for the yeast process optimisation at lab and pilot scales.     

Study of the heterogeneity of a human non-small cell bronchopulmonary epidermoid primary carcinoma: determination of the DNA content of NSCLCN6L2 cells and of cloned sub-populations

A continuous cell line, NSCLCN₆L₂, was established in vitro from a human bronchopulmonary epidermoid carcinoma and then cloned on agar gel and by selective media. The DNA content of each sub-population was compared with that of the parent line by flow cytometry. This study showed the heterogeneity of the NSCLCN₆L₂ line and the possibility of selection by cloning distinct ploidy sub-populations (group 1: diploid lines; group 2: hypotetraploid lines; group 3: a diploid and tetraploid line); it also allowed the time-course of the evolving lines to be followed. Correlation of these results with other properties of the different sub-populations will provide a better understanding of their biological behavior, particularly of their chemosensitivity.     

The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization.

The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.     

Rapid identification and isolation of zygotes and the kinetics of mating in Saccharomyces cerevisiae

Summary Flow cytometry has been used in the selection of fusion products of the yeast Saccharomyces cerevisiae. Parental cells of opposite mating type were stained with different fluorescent dyes, permitting rapid identification of zygotes from natural matings based on dual-color flow cytometry. This procedure was then used to study the kinetics of mating in yeast and the physical and biological parameters that affect these kinetics, such as cell concentration, parental ratios, and parental strain growth rate.