A novel deep red/low infrared fluorescent flow cytometric probe, DRAQ5NO, for the discrimination of intact nucleated cells in apoptotic cell populations

BACKGROUND: The linking of intracellular metabolism of anticancer drugs with cellular response is problematic. We describe a new probe for cellular integrity, based upon a structure which has the additional potential to act as a substrate for cytochrome P450-dependent bioreductive metabolism. DRAQ5NO is an N-oxide modified anthraquinone with optimal fluorescence excitation maxima compatible with He-Ne (633 nm) and Kr-Ar (647 nm) lasers. METHODS: DRAQ5NO-loading and Annexin V binding was monitored using dual-laser flow cytometry (488 nm/633 nm wavelengths) in human lymphoma cultures undergoing anticancer drug- (etoposide; VP-16) induced apoptosis. RESULTS: DRAQ5NO gave an Em(lambdamax) of 700.5 nm but retains DNA binding potential with an emission wavelength red-shift of approximately 12 nm. The agent showed reduced cytotoxicity and a limited capacity to accumulate within cells compared with the non-N-oxide form that shows a high nuclear targeting capacity in intact cells. DRAQ5NO/Annexin V provides for a positive discrimination between intact cells, membrane-compromised cells, cellular debris, and early stage apoptotic cells. CONCLUSIONS: The spectral properties of DRAQ5NO allow for the use of visible range fluorochromes and differential excitation in multilaser systems for tracking apoptotic populations with implications for the measurement of bioreductive potential in complex tumour populations simultaneously undergoing physiologically or drug-induced apoptosis.     

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4⁺ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.Abbreviations Ab antibodies - APC antigen-presenting cells - BG Birbeck granules - DNCB 1-chloro-2,4-dinitrobenzene - DNFB 2,4-dinitrofluorobenzene - DTAF dichlorotriazinylfluorescein - FSC forward light scatter - LC Langerhans cells - LSCM laser scanning confocal microscopy - MHC major histocompatibility complex - MAb monoclonal antibodies - PFA paraformaldehyde - SSC side light scatter     

Study of propidium iodide binding to DNA in intact cells by flow cytometry

We studied thein situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by “best-fitting” of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of thein situ chromatin turned out to be reduced with respect to the nonin situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.     

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.     

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.     

Monitoring reactive oxygen species formation and localisation in living cells by use of the fluorescent probe CM-H2DCFDA and confocal laser microscopy

Reactive oxygen species (ROS) develop as a consequence of wounding, light stress and chemical imbalances but act also as signals in living cells. The integrity of cells is seriously endangered, if ROS cannot be controlled by scavenging molecules and other repair mechanisms of the cell. For studying ROS development and signalling under stress, a reliable indicator is needed. We have tested the ROS sensitive dye 5-(and-6) chloromethyl-2',7' dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) using onion bulb scale and leaf epidermis as well as Arabidopsis leaves and protoplasts. ROS were generated by several fundamentally different methods—externally applied hydrogen peroxide, heat shock, high light or wounding. Confocal microscopy and fluorescence quantification over time showed that the indicator responds in an additive and dose-dependent manner. The response to externally applied hydrogen peroxide followed saturation kinetics, consistent with a channel-mediated uptake of the stressor across the plasma membrane. An inherent problem of the tested indicator was the uneven uptake in tissues, as compared with protoplasts, making it difficult to discriminate an uneven indicator distribution from an uneven ROS distribution. However, in protoplasts and under carefully designed preparation conditions CM-H₂DCFDA is a useful general ROS indicator. Subcellularly, the de-esterified probe localised to the cytosol, to mitochondria and to chloroplasts.     

A novel technique for identification of sites of anion transport in intact cells and tissues using a fluorescent probe

Abstract The purported blocker of anion transport 4, 4′ di-isothiocyano-2-2′ stilbene disulfonate (DIDS) has been shown to partially inhibit ³⁶Cl^? influx, ³⁶CIO^?₃ influx and ³⁵SO^2?₄ influx into Pisum salivum L. cv. Feltham First seedlings. This inhibitory effect could be prevented by pretreatment with the respective unlabelled medium. There was no effect of DIDS on ¹⁴C methylamine influx. The results are consistent with the hypothesis that the binding of DIDS to the site of anion-carrier interaction is responsible for its observed inhibitory effects on anion fluxes. The fluorescent properties of DIDS upon binding to membrane proteins was exploited in an attempt to examine the major sites of anion pumping in whole roots. The results show clearly that in the presence of DIDS the epidermal layers became brightly fluorescent, while cortical layers did not fiuoresce. Lycopersicum esculentum cells taken from locular fluid were plasmolysed using sucrose solution, and the patterns of fluorescence in the presence of DIDS showed in an unambiguous way that the fluorescence is associated with cell membranes. The potential usefulness of this technique to probe sites of anion transport in whole plants and tissues is discussed.     

Significant non-S-phase DNA synthesis visualized by flow cytometry in activated and in malignant human lymphoid cells

The development of a monoclonal antibody to the deoxynucleoside bromodeoxyuridine (BrdU), combined with two parameter flow cytometry, has allowed us to examine large numbers of cells for non-S-phase DNA synthesis. Three human lymphoid cell populations were studied to determine the level of deoxynucleoside (dN) incorporation as a function of DNA content. In each population, non-S-phase DNA synthesis was observed. In a rapidly growing human T-lymphoblastoid cell line (CCRF-CEM), 53% of dN incorporation occurred in G0/G1 plus G2 + M. In chronic lymphocytic leukemia (CLL) cells stimulated with tetradecanoylphorbol acetate (TPA), 45% of the observed burst in thymidine incorporation was found to be localized to G0/G1 cells. Non-S-phase incorporation was not, however, limited to neoplastic cells. Normal human peripheral blood B cells treated with the Cowan strain of Staphylococcus aureus (CSA) undergo a transient burst in thymidine incorporation, but do not go on to divide in the absence of other stimuli. Flow-cytometric analysis showed that 80% of this CSA-stimulated dN incorporation was into G0/G1 cells. These data are consistent with a more dynamic state of DNA synthesis than usually envisioned. Furthermore, the data show that although thymidine incorporation levels are related to incorporation of dN into DNA, they can be unrelated to cell proliferation.     

Cell and nuclear enlargement of SW480 cells induced by a plant lignan, arctigenin: evaluation of cellular DNA content using fluorescence microscopy and flow cytometry

Arctigenin is a natural plant lignan previously shown to induce G(2)/M arrest in SW480 human colon cancer cells as well as AGS human gastric cancer cells, suggesting its use as a possible cancer chemopreventive agent. Changes in cell and nuclear size often correlate with the functionality of cancer-treating agents. Here, we report that arctigenin induces cell and nuclear enlargement of SW480 cells. Arctigenin clearly induced the formation of giant nuclear shapes in SW480, as demonstrated by fluorescence microscopic observation and quantitative determination of nuclear size. Cell and nuclear size were further assessed by flow cytometric analysis of light scattering and fluorescence pulse width after propidium iodide staining. FSC-H and FL2-W values (parameters referring to cell and nuclear size, respectively) significantly increased after arctigenin treatment; the mean values of FSC-H and FL2-W in arctigenin-treated SW480 cells were 572.6 and 275.1, respectively, whereas those of control cells were 482.0 and 220.7, respectively. Our approach may provide insights into the mechanism behind phytochemical-induced cell and nuclear enlargement as well as functional studies on cancer-treating agents.     

Phagocytic cells in blood from rainbow trout, Salmo gairdneri (Richardson), characterized by flow cytometry and electron microscopy

An in vitro assay for estimating the proportion of phagocytic cells among peripheral leucocytes from rainbow trout by flow cytometry (FCM) and fluorescence microscopy was evaluated. Data from FCM were compared with fluorescence microscopic observations and good correlation ( r = 0.87) was found. The influence of various culture conditions, such as serum type, duration of incubation and temperature, on the in vitro phagocytic assay was investigated. Cultures supplemented with brown trout serum and incubated for 18 h at 19° C were considered to give optimal conditions for phagocytosis. The proportion of phagocytic cells detected in the peripheral blood leucocyte preparation was 3.3 ± 1.5% with FCM and 5.5 ± 2.4% with fluorescence microscopy. The applicability of the method was demonstrated in a preliminary study with arsenic. In a concentration of 1 μg ml⁻¹, arsenic increased the proportion of actively phagocytic cells, but, at a high concentration, 100 μg ml⁻¹, it decreased the phagocytic activity. Electron microscopy was used for morphological classification of the peripheral leucocytes throughout the study.     

In aged primary T cells,mitochondrial stress contributes to telomere attrition measured by a novel imaging flow cytometry assay

The decline of the immune system with age known as immune senescence contributes to inefficient pathogen clearance and is a key risk factor for many aged‐related diseases. However, reversing or halting immune aging requires more knowledge about the cell biology of senescence in immune cells. Telomere shortening, low autophagy and mitochondrial dysfunction have been shown to underpin cell senescence. While autophagy has been found to control mitochondrial damage, no link has been made to telomere attrition. In contrast, mitochondrial stress can contribute to telomere attrition and vice versa. Whereas this link has been investigated in fibroblasts or cell lines, it is unclear whether this link exists in primary cells such as human lymphocytes and whether autophagy contributes to it. As traditional methods for measuring telomere length are low throughput or unsuitable for the analysis of cell subtypes within a mixed population of primary cells, we have developed a novel sensitive flow‐FISH assay using the imaging flow cytometer. Using this assay, we show a correlation between age and increased mitochondrial reactive oxygen species in CD8⁺ T‐cell subsets, but not with autophagy. Telomere shortening within the CD8⁺ subset could be prevented in vitro by treatment with a ROS scavenger. Our novel assay is a sensitive assay to measure relative telomere length in primary cells and has revealed ROS as a contributing factor to the decline in telomere length.     

A novel fluorescent fatty acid, 5-methyl-BDY-3-dodecanoic acid, is a potential probe in lipid transport studies by incorporating selectively to lipid classes of BHK cells.

The 5-methyl-BDY-3-dodecanoic acid (B12FA) labelling of BHK cell lipids was analyzed by thin layer and reverse phase column chromatography. Incorporation to phospholipids was selective: over 90% of B12FA label was enriched in phosphatidylcholine. The major molecular species of PC was that containing palmitate as the unlabelled fatty acid. Small amounts of label was also found in other phosphoglycerides, but not in sphingomyelin. Triglycerides and diglycerides constituted the main B12FA-labelled neutral lipid classes; however, no label was found in cholesterol esters. B12FA was degraded to shorter homologues, which had significantly slower lipid incorporation rates. B12FA-labelled cells displayed in a microscope initially green reticular type fluorescence, but later red spherical structures, representing neutral lipid droplets, could also be seen. It is concluded that B12FA does not incorporate indiscriminately to all lipid classes of BHK cells, but is enriched to PC, diglycerides and triglycerides, which could be utilized in studies on lipid transport as well as metabolism.     

Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization

Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1–2 copies of HPV DNA type 16 and 10–50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1–2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells. Considered altogether our results shows that in situ hybridization is a powerful technique to detect small amounts of nucleic acid sequences but the choice of the technique for cell examination is important. Single genes of HPV were visualized most efficiently by association of FISH with LSCM or quantitative microscopy with an intensified CCD camera.     

The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique.

BACKGROUND: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death. METHODS: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay. RESULTS: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity. CONCLUSIONS: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay.     

Recovery,visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.