DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry

During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC.     

Chromosome analysis by high illumination flow cytometry

Fluorescence measurements from metaphase chromosomes of the Chinese hamster, stained with propidium iodide excited at high illumination irradiance, completely resolve each chromosome type. The measurements are performed in a specially designed flow cytometer that achieves high irradiance (4 MW/cm2) by using high power laser output (2 W at 488 nm) focused to small spot size (1% irradiance variation over 2 microns). The coefficient of variation of each chromosome peak is near 1.5%. Saturation of the fluorescence transition and photobleaching, two consequences of high irradiance, are shown to occur. Even with a nonlinear dependence of fluorescence upon illumination irradiance, fluorescence retains a proportional response to chromosome type; each chromosome peak maintains a consistent ratio to the others at every irradiance. No perturbation of fluorescence by the optical or geometrical properties of the chromosomes is evident. The advantages of high irradiance illumination are an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories on the precision. These benefits improve the resolution of chromosome analysis by flow cytometry, particularly the resolution of smaller chromosomes.     

Chromosome banding analysis by slit-scan flow cytometry

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers.     

DNA measurement and cell cycle analysis by flow cytometry

Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. Protocols for DNA measurement have been developed including Bivariate cytokeratin/DNA analysis, Bivariate BrdU/DNA analysis, and multiparameter flow cytometry measurement of cellular DNA content. This review summarises the methods for measurement of cellular DNA and analysis of the cell cycle and discusses the commercial software available for these purposes.     

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.     

Continuous measurement and analysis of staining kinetics by flow cytometry

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic or absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells. A single-channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64-channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A least-squares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants. Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time-dependent phenomena by FCM.     

Characterization of cytokine interactions by flow cytometry and factorial analysis

BACKGROUND: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS: Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS: In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS: Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.     

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial commu-nity has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophy-siological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechan-ism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating 'islands of salinity'. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the 'island-of-salinity' effect on the active microbial community in a sandy desert system.     

Carbon-isotopic analysis of microbial cells sorted by flow cytometry

One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high‐precision measurement of ¹³C in specific populations of microbial cells that are purified by fluorescence‐activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their ¹³C content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home‐built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of δ¹³C in whole cells with precision better than 0.2‰ for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 10⁴ eukaryotic or 10⁷ bacterial cells per sample. Coupled with ¹³C‐labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM‐IRMS also permits useful studies of natural abundance variations in ¹³C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.     

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial community has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophysiological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechanism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating ‘islands of salinity’. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the ‘island-of-salinity’ effect on the active microbial community in a sandy desert system.     

Cell-cycle analysis of fission yeast cells by flow cytometry

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.     

Alveolar macrophage-environmental particle interaction: analysis by flow cytometry

Inhaled particulates such as pollutant particles, allergens, and microorganisms are rapidly cleared by alveolar macrophages (AMs). Methods for analysis of AM-particle interaction have been hindered by the lack of a convenient assay. Flow cytometry offers rapid, sensitive, and reproducible measurements of single cells in suspension. Multiple parameters can be measured in real time. Here we will review the application of flow cytometry to the study and characterization of AM receptors for unopsonized environmental particles. We will discuss the role of this technique in identifying a key AM receptor system involved in lung defense. Multiparametric flow cytometry to analyze intracellular functional parameters, though a powerful and unique tool, needs to be interpreted with caution. We will also discuss the advantages and limitations of flow cytometry in analysis of AM-particle interaction.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Detection of JNK and p38 activation by flow cytometry analysis

JNK and p38 protein kinases are involved in the signal transduction of apoptotic stimulus. JNK and p38 are activated by dual phosphorylation on threonine and tyrosine residues. Different techniques such as Western blotting (WB) and confocal microscopy analysis have been developed to detect the activation by using antibodies that recognize the phosphorylated forms of both enzymes. However, these techniques are time consuming, not quantitative, and dependent on subjective interpretation. Herein, we describe a flow cytometry-based analysis to detect JNK and p38 activation. Using human primary lymphocytes and Jurkat CD4(+) T cells stimulated with PMA/ionomycin, we demonstrate activation (phosphorylation) of JNK and p38, which is further confirmed by two additional established techniques (WB and confocal microscopy). Flow cytometry analysis is shown to be more sensitive than WB to detect JNK and p38 activation, which can be quantitated and enables us to study their activation within cell populations.     

Purification and analysis of rat hematopoietic stem cells by flow cytometry

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy-1 glycoprotein, OX22 recognizes the high molecular weight form(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony-forming unit spleen (CFU-S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22-), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marrow cells were incubated with allophycocyanine-OX7 Fab' (APC-OX7 Fab') and phycoerythrin B-OX22 Fab' (Phy B-OX22 Fab'). The cells were sorted with a FACS-II instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OXu20%, OX22-. The percentage of cells in the marrow with this phenotype was found to be 0.34 +/- 0.01 (mean +/- S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30-d survival), while the number of unsorted bone marrow cells required was 1.05 X 10(6). Thus, a 350-fold purification of the HSC was realized. Although CFU-S copurified with HSC, purification of only 105-fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22- -enriched HSC population could be further divided into W3/13 dim and W3/13+ subpopulations by three-parameter immunofluorescence analysis with the use of a new optical bench arrangement.     

The analysis and interpretation of DNA distributions measured by flow cytometry

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst-33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15-20% for G1 and S phases; b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.