Showing papers in "Cytometry in 1997"
TL;DR: Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology and it is expected that flow cytometry will be the dominant methodology for necro biology.
Abstract: The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology. Cytometry 27:1–20, 1997. © 1997 Wiley-Liss, Inc.
1,146 citations
TL;DR: The data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.
Abstract: The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.
351 citations
TL;DR: Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerystrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels and the power and utility of the 8 color, 10-parameter technology is shown.
Abstract: We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems. Cytometry 29:328‐339, 1997. r 1997 Wiley-Liss, Inc.
166 citations
TL;DR: This work employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence, and realized an approximately 5-fold increase in signal to noise ratio in the direct detection of RNA target probes using flow cytometry.
Abstract: Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.
147 citations
TL;DR: Flow cytometry indicates that a form of apoptosis occurs in plants, with flow cytometric characteristics similar to those of apoptotic cells in HL-60 cells.
Abstract: An early indicator of apoptosis in mammalian cells is the loss of the phospholipid membrane asymmetry of the cell. This results in exposure of phosphatidylserine on the outer surface of the plasma membrane. This change in membrane asymmetry can be analysed using annexin V. A further feature of apoptosis, DNA breaks, can be measured by the TUNEL assay. Using flow cytometry, we have identified both of these features in HL-60 cells and by modifying the techniques for plants, we have verified that these features also occur in plant cells undergoing apoptosis. In both plant and HL-60 cells, apoptosis was induced by treatment with camptothecin (1 microM). Annexin V binding was found to be an early indicator of apoptosis, occurring prior to the detection of DNA strand breaks as monitored by the TUNEL assay. In plant cells, chromatin condensation was detected prior to the detection of annexin V. No loss in membrane integrity occurred with apoptotic cells in comparison with necrotic cells. Our findings indicate that a form of apoptosis occurs in plants, with flow cytometric characteristics similar to those of apoptosis in HL-60 cells.
143 citations
TL;DR: A completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes in each cell nucleus, and its accuracies are comparable to panels of human experts (manual).
Abstract: Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei This process is called dot counting To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd, Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc, Cupertino, CA, USA) computer After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome The slides contained lymphocytes from cultured blood We compared the results of the dot counter with manual counting Manually obtained results, published in the literature, were used as the "ground truth" For a normal specimen, 975% of cells will have two dots Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly In other words, an average of 11% has to be interactively corrected, using a monitor display The machine accuracies, after interactive correction, are comparable to panels of human experts (manual) The fully automatically obtained results are biased with respect to manual counting An error analysis is given, and different causes are discussed
133 citations
TL;DR: Flow cytometry has been used to study most of the above mentioned aspects of immunosenescence, and to establish new age-related reference values, which fit the hypothesis that a complex, unpredicted remodeling of the immune system occurs with age.
Abstract: We have been studying the immune system of healthy centenarians for many years, and they provide the best example of successful aging. They are people who have escaped major age-related diseases and reached the extreme limit of human life in good clinical condition. In most cases, histories of centenarians reveal them to be free of cancer, dementia, diabetes, cardiovascular diseases, and cataracts. Moreover, in order to reach such an advanced age, they should be equipped with well preserved and efficient immuno- and defense mechanisms, and optimal combinations of an appropriate lifestyle and genetic background. Using this approach, several paradoxes emerged as far as the immune system of centenarians is concerned, regarding: i) humoral immunity (increase in plasma immunoglobulins and nonorganspecific autoantibodies, decrease in B cell number and lack of organ-specific autoantibodies); ii) cellular immunity (well preserved number of ‘‘virgin’’ T cells, a relatively intact T cell repertoire despite a thymus involuting since puberty, increased number of cells with markers of NK activity); iii) decreased peripheral blood lymphocyte tendency to programmed cell death, associated with a well preserved mitochondria functionality and intracellular bcl-2 levels. An age-related increase in the levels of adhesion molecule present on lymphocyte plasmamembrane, accompanied by a complex reshaping of the cytokine network, must be aded to this scenario. All our data fit the hypothesis that a complex, unpredicted remodeling of the immune system occurs with age. In the present review it is underlined how flow cytometry has been used to study most of the above mentioned aspects of immunosenescence, and to establish new age-related reference values. Cytometry 27:297‐313, 1997. r 1997 Wiley-Liss, Inc.
133 citations
TL;DR: A GFP-fusion protein is employed which localizes to the cellular membrane and is retained in cells upon ethanol permeabilization without prior fixation, which allows the GFP signal to be detected in cells treated with ethanol in preparation for PI staining and cell cycle analysis.
Abstract: The simultaneous detection of the green fluorescent protein (GFP) and DNA content using propidium iodide (PI) by flow cytometry is made difficult because of the unique nature of these 2 fluorogenic reagents. For PI to enter cells efficiently and to stain DNA quantitatively, the cells must first be permeabilized; ethanol treatment is a routine method to achieve this. However, this permeabilization step causes GFP, which is normally found in the cytoplasm, to leach out of the cells. Although the use of paraformaldehyde-based fixatives allows GFP to be maintained in cells and retain its fluorescence even after ethanol permeabilization, the protocol we commonly employ results in inefficient PI staining and poor quality DNA histograms. To circumvent these difficulties, we have employed a GFP-fusion protein which localizes to the cellular membrane and as such is retained in cells upon ethanol permeabilization without prior fixation. This allows the GFP signal to be detected in cells treated with ethanol in preparation for PI staining and cell cycle analysis. This property facilitates the use of GFP as a marker for transfected cells in experiments designed to characterize the effects of ectopic expression of cellular or viral genes on cell cycle progression.
132 citations
132 citations
TL;DR: It is concluded that changes in nucleic acid stability or conformation may vary during apoptosis from one cell type to another, but mitochondrial demise may be common to all apoptotic pathways.
Abstract: We characterized the ability of six SYTO nucleic acid stains and a mitochondrial stain to resolve by flow cytometry camptothecin-induced apoptotic and non-apoptotic cells. Staining live human lymphoid B-cells showed such resolution with SYTO 11, 12, 13, 14, and 16 dyes. H9, HL-60, and Jurkat cells did not show resolution with the SYTO 12 dye, but did with the others. SYTO 15 dye did not show resolution with any cell type. RNase A treatment of fixed lymphoid B-cells strongly reduced fluorescence after staining with SYTO 12 dye; the other SYTO dyes showed little or no RNase A sensitivity. Reduced SYTO 12 fluorescence may reflect RNA breakdown during apoptosis, while decreased fluorescence of the other SYTO dyes in apoptotic cells may be due to chromosomal alterations during apoptosis. In all cell types tested, clear resolution between apoptotic and non-apoptotic cells was observed with the MitoTracker Red dye CMXRos. In double-staining experiments, cells exhibiting reduced SYTO 11 fluorescence were the same as those showing decreased CMXRos fluorescence. We conclude that changes in nucleic acid stability or conformation may vary during apoptosis from one cell type to another, but mitochondrial demise may be common to all apoptotic pathways.
129 citations
TL;DR: In this article, a method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy.
Abstract: Assessment of cell viability using methods which do not require cell culture is essential in the field of aquatic microbiology, since many bacteria known to be present in aquatic environments cannot be grown in culture. The study of bacterial biofilms, which previously needed an epifluorescent microscope, has recently been enhanced by the use of flow cytometry and confocal scanning laser microscopy (CSLM). A method based on the combination of several membrane potential related dyes, a membrane integrity dye and a redox probe was used to measure cell viability by flow cytometry and confocal laser microscopy. Rhodamine-propidium iodide (PI) double staining was used to discriminate viable from nonviable cells in CSLM observations. Membrane depolarization during E. coli and Salmonella starvation measured by DiBAC4(3) incorporation (flow cytometry and CSLM) was found to be in concordance with respiratory activity as detected by a tetrazolium salt (CTC) reduction.
TL;DR: The present study shows that the recently discovered in vitro marker of apoptosis, Annexin V meets these requirements for in vivo detection, and was able to visualize apoptotic cells derived from each germ layer in the developing mouse embryo from the whole mount level up to the ultrastructural level.
Abstract: Apoptosis is of paramount importance during embryonic development. This insight stems from early studies which correlated cell death to normal developmental processes and now has been confirmed by linking aberrant cell death patterns to aberrant development. Linking apoptosis to the phenotype of a developing organism requires spatial information on the localization of the dying cells, making in situ detection essential This prerequisite limits the tools available for such studies (1) to vital dyes, which can be detected at the whole mount level only; (2) to detection based upon apoptotic morphology by routine light microscopy and electron microscopy; and (3) to staining for apoptosis associated DNA fragmentation via, e.g., the TUNEL procedure, which marks cells in a relative late phase of apoptosis. New apoptosis markers need to be specific and should preferably detect cells early during this process. In the present study we show that the recently discovered in vitro marker of apoptosis, Annexin V meets these requirements for in vivo detection. Through intracardiac injections of biotin labeled Annexin V, a Ca2+ dependent phosphatidylserine binding protein, we were able to visualize apoptotic cells derived from each germ layer in the developing mouse embryo from the whole mount level up to the ultrastructural level. Double-labeling on paraffin sections for both this method and TUNEL revealed that cells become Annexin V-biotin labeled early during the process of apoptosis.
TL;DR: The data demonstrate that the method used for red cell lysis can have definite impact on immunophenotyping and selected methods appear to be more suitable for specific applications.
Abstract: We performed a parallel evaluation of six whole blood lysis methods comparing light scatter and quantitative fluorescence intensity based on quantitative flow cytometry, of selected lymphocyte subsets and CD34+ cells. Leukocytes prepared with FACS Lysing Solution (BDIS), Immunolyse (Coulter) and Optilyse B (Immunotech) consistently gave lower forward scatter values than those prepared with ACK (BioWhitaker), Ortho-mune (Ortho) and ImmunoPrep (Coulter). Debris, defined as CD45 negative events with the threshold off, accounted approximately 80% of all events with ACK and Ortho-mune. The other lysing methods consistently yielded less debris (approximately 50%) with Immunolyse generating only approximately 16% debris. Optilyse and FACS lyse consistently displayed the lowest percentage of lymphoid cells (CD45+/CD14-) in the three part differential. The percentage of CD3+, CD20+, CD5+, and CD16/CD56+ cells was consistent with all methods but CD4 and CD8 determinants showed inconsistent variation with ACK and Ortho-mune. In addition, the fluorescence intensity of CD14 PE and CD8 PE staining was markedly decreased on cells prepared with ImmunoPrep. Finally, the clearest separation of CD34+ cells was observed with ACK and Ortho-mune. Our data demonstrate that the method used for red cell lysis can have definite impact on immunophenotyping and selected methods appear to be more suitable for specific applications.
TL;DR: Multiparameter cell-based methods are especially well suited for elucidation in human solid tumors of the genetic evolutionary sequences that could provide a rational scientific basis for determining prognosis and for optimizing therapy in individual cancer patients.
Abstract: Human solid tumors develop multiple genetic evolutionary abnormalities as they evolve. Studies that have focused primarily on early colorectal cancer have suggested that genetic instability is a prominent feature of preinvasive disease. At least two separate mechanisms for the generation of genetic instability have been identified. The first, which involves widespread microsatellite instability in near-diploid cells, affects less than one-fifth of colon cancers. The second form of genetic instability is characterized by the development of p53 gene abnormalities that result in gross aneuploidy and multiple structural chromosomal changes. p53/aneuploidy affects most colon cancers, breast cancers, and many other solid tumors. This genetic evolutionary change commonly occurs at the interface between severe dysplasia and invasive disease. Specific post-aneuploid sequences of genetic changes that are relevant to tumor progression often involve the accumulation of multiple gain-of-function abnormalities in individual cells. The co-occurrence of Her-2/neu overexpression and EGF receptor overexpression in the same aneuploid cells defines an adeno/squamous genetic evolutionary sequence that is common to ductal breast cancers, non-small cell lung cancers, and other solid tumors. Later steps in this sequence include ras and c-myc overexpression. The neuroendocrine genetic evolutionary sequence is a separate branch of the p53/aneuploidy sequence with distinctive features that include loss of Rb and raf1 overexpression. Her-2/neu overexpression is not characteristic of this sequence; c-myc amplification/overexpression is common to both p53-associated sequences. The neuroendocrine sequence is found in small cell carcinoma of the lung and in minor proportions of other solid tumors, including breast cancer. Mutiparameter cell-based methods are especially well suited for elucidation in human solid tumors of the genetic evolutionary sequences that could provide a rational scientific basis for determining prognosis and for optimizing therapy in individual cancer patients. Cytometry 29:1–27, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.
Abstract: A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3–18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells. Cytometry 29:306–312, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: A model is suggested for the ordering of some of the terminal processes in apoptosis, with annexin V and MC540 changes trailing other events in apoptotic stages.
Abstract: Many parallel processes occur during the final stages of apoptosis. It is not clear which of these processes occur in all or most models of apoptosis and which occur only in some. In addition, the temporal relationship of these events is not always well understood. Correlated flow cytometric measurements were used to address these questions. Several models of apoptosis were studied, including thymocytes treated with dexamethasone. MOLT-4 cells treated with etoposide, U937 cells treated with anti-Fas, HL-60 cells treated with camptothecin, Raji cells grown in low serum, and aged neutrophils. All models showed a decrease in LDS-751 and fluorescein diacetate (FDA) staining, an increase in staining with dihydrorhodamine 123 (dhR123) or dihydroethidium, and an acidification of the cytoplasm. In each model, these changes were highly correlated, appearing simultaneously as multiparameter measurements. Changes in membrane status detected with merocyanin 540 (MC540) and annexin V behaved differently. A population with LDS-751 and FDA changes but without annexin V or MC540 changes could be demonstrated in some models. Several models did not show any change in annexin V binding, and HL-60 did not show a change in MC540 binding during apoptosis. The loss of cell surface antigens (CD45 and CD16) from aged neutrophils occurred in the entire LDS-751 and FDA dim population, even though other membrane changes (including the appearance of annexin V binding sites) were only apparent in a subset of these cells. These results suggest a model for the ordering of some of the terminal processes in apoptosis, with annexin V and MC540 changes trailing other events in apoptosis. These results confirm the need for caution in using a single-parameter measurement as an indicator of apoptosis for any new model being studied.
TL;DR: Two membrane potential sensitive dyes and three nucleic acid dyes were used to assess the effect of surfactants on Escherichia coli and the ability of E. coli to be stained by these probes was validated at different physiological states.
Abstract: Two membrane potential sensitive dyes (Rhodamine 123 and bis-oxonol) and three nucleic acid dyes (propidium iodide, SYTO-13, and SYTO-17) were used to assess the effect of surfactants on Escherichia coli. The ability of E. coli to be stained by these probes was validated at different physiological states. Propidium iodide was used to assess the integrity of cell envelopes. Two double staining methods based on propidium iodide with SYTO-13 and bis-oxonol with SYTO-17 were used to improve the discrimination between bacteria and micelles or aggregated particles generated by the presence of surfactants. A rapid (1 h contact time between cells and surfactants, and less than 5 min for staining and obtaining data) Rhodamine 123 flow cytometric assay was developed to assess the bactericidal effect of surfactants. Cytometry 29:58–64, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: The efficiency and yield of incorporation of fluorescent nucleotides into DNA by polymerase chain reaction (PCR) have been investigated with linear amplification with single-stranded template and single primer and dUTP attached to the fluorescent label with linkers of different lengths.
Abstract: The efficiency and yield of incorporation of fluorescent nucleotides into DNA by polymerase chain reaction (PCR) have been investigated with linear amplification (PCR with single-stranded template and single primer). In the present study, we prepared single-stranded templates with defined sequences and used dUTP attached to the fluorescent label with linkers of different lengths. Incorporation and yield of the modified dUTP were reduced when the sequence demanded that multiple dyes be inserted at adjacent sites. The interactions between the polymerase and cyanine-labeled sites on the extending strand probably terminated the chain extension. Thus, because labeling density was increased, the yield of PCR was reduced. We also found that the interactions between the primer and dye-labeled sites on template disturb primer annealing and lead to a decrease in PCR yield.
TL;DR: Multiparameter flow cytometric analysis of these breast carcinomas with MAbs Do1 and Do7 showed intratumor heterogeneity for p53 accumulation, which was independent of DNA index heterogeneity.
Abstract: p53 immunostaining of histological sections shows inter- and intratumor variability in distribution and staining intensity which are usually scored semiquantitatively. In order to investigate the variation in p53 expression more accurately and its possible relation to other cellular parameters (e.g., DNA content), we have studied the possibility to measure p53 accumulation by multiparameter flow cytometry. To this end we have evaluated seven, commercially available, monoclonal antibodies (MAbs) against p53 (MAbs 1801, 240, 246, 421, 1620, Do1, and Do7) on five tumor cell lines with known p53 gene status: MCF-7 (wild-type p53 gene), COV362.cl4 and T47d (mutated p53 genes), and SAOS-2 and HL60 (no p53 mRNA). Localization of immunofluorescence was investigated with confocal laser scanning microscopy, immunofluorescence signal intensity with flow cytometry, and antibody specificity with Western blotting. Subsequently, single cell suspensions from two breast carcinomas were flow cytometrically analyzed after triple staining for p53, cytokeratin 8/18, and DNA, and compared to immunohistochemical staining. MAbs Do1 and Do7, and to a lesser extent MAb 421, accurately discriminated p53 positive from p53 negative cell lines. Even at high concentrations these MAbs yielded nuclear immunofluorescence, whereas with MAbs 1801, 240, and 246 strong cytoplasmic signals in both the p53 accumulating and p53 negative cell lines were seen. By using lower antibody concentrations the cytoplasmic immunofluorescence disappeared, but simultaneously the nuclear p53 immunostaining intensity in p53 accumulating cell lines decreased, resulting in false negative nuclei. With MAb 1620 only weak intranuclear spots were obtained in all cell lines tested. Western blotting yielded results with MAbs 1801, Do1, and Do7 in the 53 kD region of the p53 accumulating cell lines. The signal intensity obtained with MAb 1801 was much less compared to MAbs Do1 and Do7. Although all three MAbs are also described as wild-type p53 specific, only MAbs, Do1 and Do7 showed bands in the 53 kD region of cell line MCF-7. With MAb 1801 ascites and MAb 1801 supernatant an additional approximately 80 kD band was present in all cell lines tested, including SAOS-2, indicating cross reactivity of this MAb. Immunohistochemical staining of two clinical breast carcinomas confirmed the results obtained in the cell lines. Multiparameter flow cytometric analysis of these breast carcinomas with MAbs Do1 and Do7 showed intratumor heterogeneity for p53 accumulation, which was independent of DNA index heterogeneity. We conclude that MAbs Do1 and Do7 enable quantitative analysis of p53 accumulation in a multiparameter flow cytometric analysis.
TL;DR: University of Texas, Galveston, Texas, is the first university in the United States to train a post-operative oncologist to provide on-the-spot diagnosis of central giant cell carcinoma.
Abstract: Bruce H. Davis,1* Kathy Foucar,2 Wlodek Szczarkowski,3 Edward Ball,4 Tom Witzig,5 Kenneth A. Foon,6 Denise Wells,7 Pat Kotylo,8 Rebecca Johnson,9 Curtis Hanson,5 and David Bessman10 1William Beaumont Hospital, Royal Oak, Michigan 2University of New Mexico, Albuquerque, New Mexico 3Cytometry Associates, Brentwood, Tennessee 4University of Pittsburgh, Pittsburgh, Pennsylvania 5Mayo Clinic, Rochester, Minnesota 6Markey Cancer Center, Lexington, Kentucky 7Hematologics, Seattle, Washington 8Indiana University, Indianapolis, Indiana 9Berkshire Medical Center, Pittsfield, Massachusetts 10University of Texas, Galveston, Texas
TL;DR: A simple and rapid laser scanning device for solid phase cytometry that is able to detect rare cellular events at a frequency of 10(-7) in 3 min and show a linear relationship between the amount of fluorescein coupled to the cells and the fluorescence signal of the cells detected is described.
Abstract: We describe a simple and rapid laser scanning device for solid phase cytometry. This system can detect and count fluorescent cells over a 22 mm diameter surface (membrane or glass circle) as well as quantify the fluorescence that they emit. A comprehensive discrimination package includes optical and software parameters and accurately distinguishes between valid signals (e.g., labelled cells) and nonspecific signals (e.g., auto-fluorescent particles and debris). Any event detected may also be easily confirmed by visual observation after transfer of the sample to an epifluorescence microscope fitted with a motorized stage driven by the system.
We show a linear relationship between the amount of fluorescein coupled to the cells and the fluorescence signal of the cells detected. This approach is not destructive and further characterization of the sample may be carried out. We have been able to detect rare cellular events at a frequency of 10−7 in 3 min. Potential applications include monitoring of residual disease in oncology and detection of virus-infected cells circulating at very low frequencies. Cytometry 27:336–344, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition.
Abstract: In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters
TL;DR: A rapid denaturation test is developed to assess the quality of the preparations without hybridization and quantitative image analysis criteria for assuring the day-to-day quality of CGH experiments, including sensitivity, specificity, and dynamic range.
Abstract: With the recent rapid expansion in the use of the comparative genomic hybridization (CGH) technique, increased attention to quality control is essential. In the present study, we show that despite optimization and standardization of metaphase preparation techniques and the commercial availability of metaphase spreads, batch-to-batch variability of the preparations remains a significant problem. To facilitate reliable CGH analysis despite this variability, we have developed a rapid denaturation test to assess the quality of the preparations without hybridization and quantitative image analysis criteria for assuring the day-to-day quality of CGH experiments, including sensitivity, specificity, and dynamic range. Monitoring the dynamic range of the hybridizations was found to be particularly critical for achieving sensitive and reliable CGH results. This reliability can be achieved, for example, by hybridization of a green-labeled normal male DNA against red-labeled female DNA and monitoring of the green:red ratio of the X chromosome in relation to that of the autosomes.
TL;DR: This paper describes a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension, and observed that aggregate size and development kinetics depend on the culture conditions used.
Abstract: No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.
TL;DR: The assessment of the quantitative expression of cell-adhesion molecules and growth-factor receptors together with cell cycle studies of mast cells could be of value for monitoring therapy and predicting clinical outcome in aggressive SMCD.
Abstract: The immunophenotypic characteristics of both bone marrow (BM) and peripheral blood (PB) mast cells (MC), from a patient suffering from an aggressive systemic mast cell disease (SMCD), were sequentially analyzed by flow cytometry using direct immunofluorescence. Analysis was carried out at diagnosis, during clinical response induced by interferon alfa-2b/prednisone therapy, and later at relapse. Our results show that together with the CD117 and IgE characteristic markers, at diagnosis BM MC showed strong expression of CD11c, CD13, CD29, CD33, CD44, CD45, CD63, and CD71, and they were also positive for CD2, CD22, CD25, and CD54 although at a lower level. PB MC displayed similar immunophenotypic characteristics although they had a lower expression of CD11c, CD25, CD33, CD63, CD69, and CD71 with a higher reactivity for CD117. Unlike BM MC, PB MC were weakly positive for CD41a and CD61. Sequential studies showed decreased numbers of both BM and PB MC during clinical response associated with a higher expression of the CD29 and CD54 adhesion molecules. In turn, clinical relapse was related to increased numbers of PB and BM MC together with lower CD2, CD11c, CD45, and CD54 expression and a higher reactivity for the CD117 and CD25 antigens. CD2 had become negative at the last follow-up study. In addition, an increased proportion of S-phase MC was observed at relapse. These findings suggest that the assessment of the quantitative expression of cell-adhesion molecules and growth-factor receptors together with cell cycle studies of mast cells could be of value for monitoring therapy and predicting clinical outcome in aggressive SMCD. Cytometry 30:98‐102, 1997. r 1997 Wiley-Liss, Inc. Key terms: systemic mast cell disease; immunophenotype; flow cytometry; mast cells
TL;DR: It is concluded that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h and if these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.
Abstract: Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose γ-irradition prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change. Cytometry 29:242–249, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: Application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.
Abstract: Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.
TL;DR: Using three color flow cytometry, anti-human antibodies are screened for crossreactivity with macaque cells in order to determine the distribution of functionally important lymphocyte subsets in blood, lymph nodes (LN), and spleen.
Abstract: Rhesus macaques are invaluable experimental animals in biomedical research. Using three color flow cytometry, we screened anti-human antibodies for crossreactivity with macaque cells in order to determine the distribution of functionally important lymphocyte subsets in blood, lymph nodes (LN), and spleen. NK-cells are almost completely absent in LN. The percentage of B-cells expressing CD80, CD86, and the level of expression of CD20 is higher in blood than in LN. In contrast, a higher proportion of B-cells in LN stains positive for CD21 and CD35. Whereas the number of CD29hi expressing T-cells is lower, CD69 is expressed on more T-cells in LN than in blood. About one-third of CD8+ T-cells in blood are CD28-, a subset with a unique pattern of antigen expression which cannot be found in LN. In contrast to humans, a relatively high proportion of T-cells in blood also express the co-stimulatory molecules CD80 and CD86. With increasing age, the proportion of B-cells in blood declines, whereas the percentage of T-cells rises. In addition, the proportion of CD29hi expressing T-cells increases among both the CD4+ and CD8+ subsets.