Flow cytometry cell distribution

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

More recent investigations on the antimycobacterial activity of pamamycin-607 (lb) on 25 independent M. tuberculosis clinical isolates (either susceptible, mono-, or multiresistant to the first line antituberculous drugs) established minimum inhibitory concentrations MICjoo in the range of 1.5-2.0 pg/ml, while the MICjoo of lb for a bioluminescent laboratory strain of M. tuberculosis (H37Rv) was determined as 0.55 pg/ml [3a]. Parallel studies on the effect of lb on the cell cycle distribution of human (HL-60) cells by flow cytometry indicated no... 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

Flow cytometry is a very versatile technique [223] which allows the analysis of more than 104 cells per second [369,370]. This high number results in statistically significant data and distributions of cell properties. Therefore, flow cytometry is a key technique to segregate biomass (into distinct cell classes) and to study microbial populations and their dynamics, specifically the cell cycle [76, 87, 116, 200, 214, 221, 295, 329, 330, 409, 418]. Individual cells are aligned by means of controlled hydrodynamic flow patterns and pass the measuring cell one by one. One or more light sources, typically laser(s), are focused onto the stream of cells and a detection unit(s) measure(s) the scattered and/or fluorescent light (Fig. 24). Properties of whole cells such as size and shape can be... 

Flow cytometry [141, 142] is a technique that allows the measurement of multiple parameters on individual cells. Cells are introduced in a fluid stream to the measuring point in the apparatus. Here, the cell stream intersects a beam of light (usually from a laser). Light scattered from the beam and/or cell-associated fluorescence are collected for each cell that is analysed. Unlike the majority of spectroscopic or bulk biochemical methods it thus allows quantification of the heterogeneity of the cell sample being studied. This approach offers tremendous advantages for the study of cells in industrial processes, since it not only enables the visualisation of the distribution of a property within the population, but also can be used to determine the relationship between properties. As an example, flow cytometry has been used to determine the size, DNA content, and number of bud scars of individual cells in batch and continuous cultures of yeast [143,144]. This approach can thus provide information on the effect of the cell cycle on observed differences between cells that cannot be readily obtained by any other technique. 

The issue of distribution in the population of a monoseptic culture is only recently getting more attention. Studies on the single-cell level by image analysis (see the chapter in this volume by M.N. Pons), and by flow cytometry (see [4,5] and the section in the chapter by Sonnleitner in this volume), reveal that most microbial cultures are by no means a mass of identical cells, even those which do not show morphological inhomogeneity. Some of the methods discussed here can also give information on distribution of subpopulations, at least when these differ by clear-cut boundaries (for an overview see Table 2). 

Jones PGV, Haq SM (1963) The distribution of Phaeocystis in the Eastern Irish Sea. J Cons Int Explor Mer 28 8-20 Jonker R, Groben R, Tarran G, Medlin L, Wilkins M, Garcia L, Zabala L, Boddy L (2000) Automated identification and characterisation of microbial populations using flow cytometry the AIMS project. Sci Mar 64 225-234 Kamermans P (1994) Nutritional value of solitary cells and colonies of Phaeocystis sp. for the bivalve Macoma balthica (L). Ophelia 39 35-44... 

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

Based on the experimental investigation of the infection status of cells by measuring immrmofluorescence of intracellular viral proteins with flow cytometry [3] mathematical models are required, which are able to describe distributed populations of cells with different degrees of infection. For this purpose, in the present paper an internal coordinate is introduced to quantify the degree of infection and the previous approach by Mohler et al. [4] is extended accordingly. 

An alternative methodology that can be used to rapidly detect and quantitate DNA strand breaks in individual cells undergoing apoptosis utilizes flow cytometry (8). This has the advantage of simultaneous detection of the DNA strand breaks and cell-cycle distribution in individual cells. In addition this methodology has been used to detect DNA strand breaks in clinical samples before and after treatment with cytotoxic drugs (8,9). 

Blair OC, Carbone R, Sartorelli AC. Differentiation of HL-60 promyelocytic leukemia cells Simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry. Cytometry 1986 7 171-7. 

Key words Flow cytometry, DNA, Fluorescent distribution, Cell cycle... 

---