Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells

Summary The cationic lipophilic dye Rhodamine 123 (Rh123) is selectively enriched in mitochondria in a membrane potential-dependent manner. Application of drugs which interfere with the electron flow of the respiratory chain lead to a severe reduction of mitochondrial dye uptake. In this communication we show that the same effect is observed after Rh123-staining of respiratory-deficient yeast mutants. Based on this observation we used flow cytometry to discriminate respiratory-compentent and respiratory-deficient yeast cells. Combined with a cell sorter we were able to selectively enrich respiring and non-respiring yeast cells, repectively, from a mixture of cells.     

Detection of dead cells and measurement of cell killing by flow cytometry

A flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell within a large, heterogeneous population. The modern commercially available analytical instruments, which can be found in most hospitals, pathology laboratories, and cell biology research laboratories in the industrially developed countries, can now routinely measure fluorescence simultaneously at four different wavelengths, in addition to light scatter in two directions, at rates of thousands of cells per second. Thus, flow cytometry provides a powerful and versatile approach to the measurement of cell death and cell killing. However, it is important to stress that the biological nature of the phenomenon under investigation will determine which assay is appropriate, and at what stage in the process of cell killing it should be applied. This consideration is particularly important in the study of drug- or cell-mediated cytotoxicity.     

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.     

Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry

At present there is much interest in the cell tropism and host range of influenza viruses, especially those of the H5N1 subtype. We wished to develop a method that would enable investigation of attachment of infectious virus through the interaction of the hemagglutinin molecule and live mammalian and avian cells and the subsequent infection of these cells. To this end, influenza viruses of various HA subtypes were constructed that either carry the green fluorescent protein (GFP) instead of the neuraminidase protein, or that express GFP in the cytoplasm of infected cells. The HA genes were derived from influenza viruses A/PR/8/34 (H1N1), A/Netherlands/178/95 (H3N2) and A/Vietnam/1194/04 (H5N1). Using these pairs of viruses, attachment and post-attachment events in the virus replication cycle can be distinguished. In general, the expression of NeuAc(alpha2-3)Gal or NeuAc(alpha2-6)Gal receptors on the cells tested corresponded with the attachment of the viruses that were studied with respect to predicted receptor specificity. Virus attachment was not always predictive for efficient infection of the cells.     

Flow cytometry: an 'old' tool for novel applications in medical genetics

Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. In the meantime, flow cytometers have become user-friendlier, less expensive instruments with an increasing importance in clinical diagnostics. Besides the classical fields of application, such as immunophenotyping blood cells or analyzing the cell cycle status by measuring the DNA content, novel flow cytometric methods have been developed to identify and to quantify disease-related gene sequences. Here we give an overview of current and future applications, including the detection of viral sequences via microsphere-based PCR assays and the analysis of single nucleotide polymorphisms, reflecting individual phenotypic traits. Furthermore, flow cytometry allows the quantification of gene expression changes as well as the isolation of differentially expressed gene sequences. Flow cytometry is also convenient for multiplex analyses, e.g. when hybridizing DNA samples to a mixture of various microsphere populations each coated with different DNA probes. Last but not least, the use of magnetic beads in combination with flow cytometers coupled with automated devices enables molecular diagnostics on a large scale. Overall, this review demonstrates flow cytometry as a rapid, sensitive, and reproducible tool applicable to a wide range of medical genetic approaches.     

Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue: an additional diagnostic tool in surgical pathology

A major drawback of immunohistochemical detection of monoclonality in B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used. Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10). Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material. In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (kappa and lambda) was performed. This experiment showed that this cross-linking was minimal (less than 2%). All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-cell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues.     

Dual‐labelled antibodies for flow and mass cytometry: A new tool for cross‐platform comparison and enrichment of target cells for mass cytometry

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross‐platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal‐labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome‐conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR^® labelling protocol. After labelling with ¹⁴¹Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4⁺ T‐cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19‐VioBlue‐¹⁴²Nd, CD20‐VioGreen‐¹⁴⁷Sm, CD27‐Cy5‐¹⁶⁷Er and CD38‐Alexa488‐¹⁴³Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.     

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.     

Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo

The goal of this work is to develop in vivo photoacoustic (PA) flow cytometry (PAFC) for time-resolved detection of circulating absorbing objects, either without labeling or with nanoparticles as PA labels. This study represents the first attempt, to our knowledge, to demonstrate the capability of PAFC with tunable near-infrared (NIR) pulse lasers for real-time monitoring of gold nanorods, Staphylococcus aureus and Escherichia coli labeled with carbon nanotubes (CNTs), and contrast dye Lymphazurin in the microvessels of mouse and rat ears and mesenteries. PAFC shows the unprecedented threshold sensitivity in vivo as one gold nanoparticle in the irradiated volume and as one bacterium in the background of 10(8) of normal blood cells. The CNTs are demonstrated to serve as excellent new NIR high-PA contrast agents. Fast Lymphazurin diffusion in live tissue is observed with rapid blue coloring of a whole animal body. The enhancement of the thermal and acoustic effects is obtained with clustered, multilayer, and exploded nanoparticles. This novel combination of PA microscopy/spectroscopy and flow cytometry may be considered as a new powerful tool in biological research with the potential of quick translation to humans, providing ultrasensitive diagnostics of pathogens (e.g., bacteria, viruses, fungi, protozoa, parasites, helminthes), metastatic, infected, inflamed, stem, and dendritic cells, and pharmacokinetics of drug, liposomes, and nanoparticles in deep vessels (with focused transducers) among other potential applications.     

Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry

A molecular probe was developed to monitor caspase activity in living cells by flow cytometry. It consists of CFP and YFP with a peptide linker containing two caspase-cleavage sites (LEVD). Its expression resulted in intense fluorescence resonance energy transfer (FRET), whereas cleavage of this linker by caspases eliminated FRET because of physical separation of the CFP and YFP moieties. Using flow cytometry, cells expressing this probe exhibited two patterns, strong FRET and diminished or absent FRET. The appearance of diminished FRET was inhibited by a pan-caspase inhibitor z-VAD or D->A mutations in the LEVD sequence and was markedly increased by apoptosis-inducing agents, etoposide and camptothecin, or overexpression of a caspase 8-red fluorescent protein fusion protein. Importantly, this probe's ability to monitor caspase activity was comparable with results obtained with fluorogenic substrates or fluorochrome-labeled inhibitors of caspases. Specific caspase inhibitors indicated the probe was highly sensitive to cleavage by caspase 6 and 8, less sensitive to caspase 4, and resistant to other caspases. Activation of caspase 8 by Fas engagement markedly increased the probe's cleavage, whereas treatment of caspase 8-deficient cells with anti-Fas did not increase cleavage. However, staurosporine induced cleavage of the probe in caspase 8-deficient cells by a mechanism that was inhibited by overexpression of bcl-x. Taken together, the data indicate that this caspase-sensitive probe can be used to monitor the basal and apoptosis-related activities of caspases, including an initiator caspase, caspase 8, and effector caspases, such as caspase 6.     

Microspheres, nanospheres and flow cytometry: from cellular to molecular analysis

Micro- and nanospheres are tightly associated with the development of flow cytometry. They are indispensable tools to optimize diffraction and fluorescence signals as well as for fluorescence calibration and cellular purification (magnetic micro- and nanospheres). They are also usefull to evaluate phagocytosis and to detect slightly expressed antigens. Recently, developments of microspheres-based flow cytometric assays have raised to quantify soluble analytes in biological fluids, cellular and tissue samples. The technology utilizes spectrally distinct fluorescent microspheres as a solid support for a conventional immunoassay, affinity assay or DNA hybridisation assay which is subsequently analyzed on a flow cytometer. Several multiplexed bead systems are now available facilitating the development of multiplexed assays that simultaneously measure many different analytes in few microliters of sample. Some recent applications with fluorescent microspheres coated with antibodies or oligonucleotides include cytokines and PCR products quantitation and single nucleotide polymorphism genotyping. Thus, multiplex assays using microspheres and flow cytometry technologies are exciting techniques which have the potential to contribute to the development of efficient diagnostic and research methods.     

Utility and limitations of flow cytometry DNA content analysis in neoplastic cells]

A review of the literature concerning the analysis of nuclear DNA content by flow cytometry is made and it is compared with own experience. The advantages and the limits of this technique are examined. The practical problems in the interpretation of the histograms and the value of the measurements are discussed. The importance of this analysis in diagnosis and in staging of many tumors and the clinical involvements are emphasized. In many tumors DNA ploidy represents a new independent prognostic variable that is useful to separate the cases with a potential worse behaviour in an early stage, when other classic parameter are not available. It is also stressed the importance of flow cytometric DNA analysis of tumor cells. This is made on the same tissues that pathologists use for histopathological diagnosis and the results are important in many cases for a diagnostic accuracy.     

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry.

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10 ng/ml TNF-alpha for 4 h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2+/-0.1 to 5.8+/-0.1 and the lysosomal increased from 4.3+/-0.4 to 5.2+/-0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.     

Lymphocyte immunophenotyping in an elderly population: age, sex and medication effects--a flow cytometry study

Flow cytometry studies of 179 individuals aged 55-95 years reveal correlations of various hematological and immune cell measures (quantification of leukocytes, leukocyte subtypes, platelets, and erythrocytes) with age, sex, common medications (aspirin, ulcer therapy, estrogen replacement therapy), and health history variables (minor arthritis, allergy, smoking, stress and exercise). Possible reasons for some of these findings are discussed.     

Immunofluorescence and flow cytometry analysis of fibronectin and laminin binding to Sporothrix schenckii yeast cells and conidia

The adherence of Sporothrix schenckii yeast cells to several extracellular matrix (ECM) components has already been demonstrated, but the mechanisms of these interactions remained to be defined. In indirect immunofluorescence assays with polyclonal antibodies directed towards the ECM proteins, both hyphae and yeast cells of S. schenckii exhibited the ability to bind laminin and fibronectin. Flow cytometry confirmed the binding of these proteins, and revealed a significant greater binding capability for the yeast cells than for the conidia. Fibronectin and laminin binding was dose-dependent and specific. In addition, competition experiments with synthetic peptides mimicking the adhesive sequences of these proteins, or with cell wall fractions and carbohydrates constitutive of their sugar chains, were performed in order to specify the peptide or carbohydrate motifs involved in the recognition process. A 50% reduction was noticed in fibronectin binding in the presence of the synthetic peptide RGD, and a 38% reduction in laminin binding with the peptide YIGSR. Some carbohydrate-containing fractions of the yeast cell wall also inhibited the binding of fibronectin, but had no significant effect on laminin binding. Together, these results suggest the presence at the yeast surface of distinct receptors for laminin and fibronectin.     

Use of propidium iodide staining and flow cytometry to measure antibody-mediated cytotoxicity: Resolution of complement-sensitive and resistant target cells

Propidium iodide uptake as an indicator of cell viability was used to measure antibody-mediated cytotixicity. In conjunction with analysis using the fluorescence-activated cell sorter, this method is as sensitive as isotope release assay systems and has the advantage of gathering multiparameter data on individual cells. This feature enables the resolution of antibody-binding target cells into a population which is sensitive to complement-mediated killing and another population which is resistant to complement-mediated killing.