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Results for “Laser Scanning Cytometry”

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PubMed1994

Evaluation of a new slide-based laser scanning cytometer for DNA analysis of tumors. Comparison with flow cytometry and image analysis.

DNA measurements generated by a new automated slide-based cytometer, the laser scanning cytometer (LSC), were compared with those produced by commercial flow cytometry (FCM) and image analysis (IA) devices. Laser scanning-cytometric analysis was performed by scanning alcohol-fixed, propidium iodide-stained tumor imprints with a 5-microns spot laser beam. Fifty-three malignant tumors (51 breast carcinomas and 2 lung carcinomas) were studied. Ploidy concordance rates for FCM versus LSC, IA versus LSC, and FCM versus IA were 96%, 91%, and 91%, respectively. Statistically significant agreement between methods was determined by linear regression analysis of DNA indices. Synthesis-phase fractions generated by FCM and LSC also were comparable, as demonstrated by linear regression (r = .83). Mean coefficients of variation for the LSC compared favorably with those for FCM and IA. The few discrepancies in ploidy status between methods could be explained by sampling error, the presence of possible near-diploid aneuploid populations that could not be effectively resolved by one or another modality, and the visual selection bias with IA when small aneuploid cell populations were present. The LSC shares many useful features with FCM, including automation, accuracy of quantitation, rapidity, and generation of reliable information regarding cell proliferation (synthesis-phase fraction). In addition, it has some of the advantages of IA, such as minimal tissue requirement, no need for special preparation, and the potential for visual selection of the cells measured. The LSC holds great promise for use in the clinical laboratory because of these combined characteristics.

Breast Neoplasms↗

PubMed1999

Assessment of DNA content in formalin-fixed, paraffin-embedded tissue of lung cancer by laser scanning cytometer.

A new cytometric device, a laser scanning cytometer was developed to overcome the limitations of flow cytometry (FCM) and image analyses. The purpose of this study was to develop a method that allows laser scanning cytometry (LSC) to be used for measuring the cellular DNA content of paraffin-embedded tissues. Paraffin-embedded lung cancer tissue from 30 patients was analyzed by both FCM (p-FCM) and LSC (p-LSC). In addition, touch preparations from fresh frozen tissues were prepared to provide material for LSC (f-LSC). The limits of agreement for the DNA indices (DI) measured by p-LSC and p-FCM were -0.07 to 0.07, indicating that for a given case, these methods would be expected to differ by no more than 0.07. The limits of agreement for comparisons between the other materials and methods were wider and depended upon the size of the measurements. Agreement between f-LSC and p-FCM was good for small DI values, but poor for large values. Agreement between f-LSC and p-LSC was poor for small and large DI values, but good for moderately sized values. Discordancies in DNA ploidy status between different materials and methods may have been caused by either the heterogeneity within tumors, sampling errors or differences in the interpretation of histograms. This method allows a comparison of the results of DNA analysis with histologic findings from hematoxylineosin-stained sections and the prognosis of the patients.

Aneuploidy↗

PubMed2000

Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation.

BACKGROUND: Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation. MATERIALS AND METHODS: Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry. RESULTS: The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation. CONCLUSIONS: These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.

Annexin A5↗

PubMed1999

Mast cells express connexins on their cytoplasmic membrane.

BACKGROUND: Because of the close association between mast cells and fibroblasts in the microenvironment and the importance of connexins (Cxs) in fibroblast communication with other cells, we hypothesized that mast cells also express Cxs, allowing them to similarly communicate with other cells through gap junctions. OBJECTIVES: We sought to identify the expression of Cxs (particularly Cx43, Cx32, and Cx26) by murine mast cells. METHODS: The expression of Cxs was studied by RT-PCR, Northern blot analysis, Western blot analysis, flow cytometry, and confocal laser scanning microscopy. RESULTS: In this report we demonstrate that murine bone marrow cultured mast cells and the growth factor-independent murine mast cell line C57, express Cx43 and Cx32 as assessed by RT-PCR, Northern blot analysis, Western blot analysis, and flow cytometry, but do not express Cx26. We also show, by confocal laser scanning microscopy, that Cx43 localizes to the cytoplasmic membrane of mast cells in a pattern similar to that seen in fibroblasts. CONCLUSIONS: Mast cells express Cx43 and Cx32, and Cx43 is associated with the cytoplasmic membrane, suggesting that mast cells have the potential to communicate with other cells in their microenvironment in part through gap junctions.

Animals↗

PubMed1997

Assessment of E. coli and Salmonella viability and starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4(3), propidium iodide, and CTC.

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.

Barbiturates↗

PubMed2006

An improved method for the selective detection of fungi in hospital waters by solid phase cytometry.

Yeast cells and mould spores can be fluorescently labelled with the viability stain carboxyfluorescein diacetate (CFDA) and detected on a membrane filter by laser scanning (solid phase cytometry, SPC). Although the selectivity of an existing commercial SPC procedure for fungi is ensured by using a 2 microm pore size membrane filter and a pre-incubation on a proprietary spore swelling/activation medium, some bacteria are still co-detected. In the present study, the selectivity for fungi has been enhanced by combining the green fluorescent CFDA with a second red fluorescent label, i.e. TRITC-concanavalin A, targetting fungal but not commonly bacterial cells. Additional improvements resulted from the prolongation of the pre-incubation and from the extra-rinsing of the membrane filter. The improved method was applied to detect fungi in hospital waters, dialysis fluids and endoscopic rinse waters. In general, SPC detected more fungi in water than plate methods. The occurrence of fungi in dialysis fluid and endoscopic rinse water was rare. Evidence for the presence of fungal viable but non-culturable (VBNC) cells in water was weak.

Colony Count, Microbial↗

PubMed2005

[Slide-based multi-parametric cytometry in ENT. Perspectives for the clinic and research].

BACKGROUND: Flow cytometry is the standard method for the multi-parametric analysis of cells. However, for about a decade, an instrument has been available which analyses fluorescing cells immobilised on slides called a laser scanning cytometer (LSC). Its design, according to the principles of slide-based cytometry, promises many advantages, especially in the analysis of minimal sample volumes. METHODS AND PATIENTS: To date, applications for cultured cells and animal models have been established. Its use for clinical purposes, however, remains to be critically evaluated. We analysed a variety of specimens obtained in our clinical routine. RESULTS: First, the instrument's resolution was evaluated using standardised particles. This showed a very good sensitivity across a wide range of fluorescence intensities at various wavelengths. Next, diverse applications for tissue engineering, immunophenotyping, and ENT-oncology were tested. Considering its microanalytical capacities, LSC proved to be a convincing tool for clinical use. Additionally, complex structures such as bi-layers of cultured cells were analysed. CONCLUSION: A broad spectrum of applications in clinical practice and research for the LSC is evident.

Equipment Design↗

PubMed1991

Cell cycle-specific expression and nuclear binding of DNA polymerase alpha.

The expression and distribution of DNA polymerase alpha was measured by cytometry and confocal laser scanning microscopy. Expression was proportional to DNA content in proliferating cells, while only S-phase cells retained DNA polymerase alpha after detergent extraction. Nuclear DNA polymerase alpha binding may be one of the key events of S-phase entry.

Animals↗

PubMed2002

B cells express intracellular but not surface receptors for testosterone and estradiol.

Increasing evidence indicates the existence of membrane receptors for testosterone (mAR) and estradiol (mER) on the surface of cells, besides the classic intracellular androgen receptor (iAR) and estrogen receptors (iER). Here, we investigate the occurrence of sex steroid receptors in B cells isolated from the spleen of C57BL/10 mice using magnetic cell sorting. RT-PCR reveals the presence of iAR, iERalpha, but not iERbeta. Using different anti-iAR and anti-iER antibodies flow cytometry and confocal laser scanning microscopy (CLSM) localize iAR and iERalpha in the cytoplasm, which are translocatable to the nucleus upon incubation with testosterone (T) and 17beta-estradiol (E(2)). The surface of B cells is devoid of iAR and iERalpha and does not bind any T and E(2) conjugated to BSA-FITC as revealed by flow cytometry and CLSM. In accordance, T and E(2) are not able to induce any rapid rise in in the intracellular free Ca2+ concentration of Fura-2 loaded B cells. Our data indicate that B cells express neither mAR nor mER on their surfaces, in contrast to other major cells of the immune system such as T cells and macrophages.

Animals↗

PubMed2000

Specific binding of recombinant Listeria monocytogenes p60 protein to Caco-2 cells.

The Listeria monocytogenes p60 is a major extracellular protein, which is believed to be involved in the invasion of these bacteria into their host cells. So far the mechanism by which p60 participates in the internalization or penetration of L. monocytogenes is still veiled. To determine the possibility of a direct interaction of p60 with the host cell surface, the iap gene was recombinantly expressed in Escherichia coli and used for binding studies with the enterocyte-like Caco-2 cells. Fluorescence activated flow cytometry and confocal laser scanning microscopy revealed a cell membrane specific staining with p60, which implications in Listeria virulence are discussed.

Bacterial Proteins↗

PubMed2004

On the biological activity of anti-ICAM-1 oligonucleotides complexed to non-viral carriers.

An important challenge in antisense technology remains the adequate delivery of the oligonucleotides (ON) to individual cells. Understanding the subcellular distribution of ONs and their carrier is essential to explain the (lack of) biological activity. The ability of several cationic carriers to efficiently deliver anti-ICAM-1 oligonucleotides to their site of action was studied using a cell-based assay. In this assay we evaluated the ability of the ONs to downregulate the expression of the ICAM-1-protein in A549 cells. To understand why some carrier/ONs combinations showed biological activity while others failed, flow cytometry and confocal laser scanning microscopy (CLSM) measurements were used to study cellular uptake and intracellular distribution of the (fluorescently labeled) ONs. We showed that free ONs (both PS-ONs and PO-ONs) and ONs complexed to pEGpEI failed to decrease the ICAM-1 protein level. This was due to the inability of the (free or complexed) ONs to enter the cell, as shown by flow cytometry and CLSM. Flow cytometry and CLSM showed cellular uptake when PO-ONs and PS-ONs were complexed to graft-pDMAEMA and Lipofectin. However, while the uptake and intracellular localization seemed similar for ONs complexed to, respectively, graft-pDMAEMA and Lipofectin, the biological activity of the ONs was clearly dependent on their carrier: both PO-ONs and PS-ONs complexed to graft-pDMAEMA reduced the ICAM-1 expression; however, when complexed to Lipofectin only PS-ONs showed biological activity. Also, PS-ONs complexed to graft-pDMAEMA were more active than PO-ONs complexed to graft-pDMAEMA which could not be explained by the results from CLSM and flow cytometry. While the ICAM-1 assay proves whether a certain pharmaceutical carrier successfully delivers ONs or not, it does not answer the important question why one carrier is successful while another one fails. Also, our study shows that flow cytometry and CLSM, although useful techniques, failed to clearly explain the difference in transfection behavior between graft-pDMAEMA and Lipofectin. As ONs become susceptible to degradation by cytosolic DNase as soon as they are released from their carrier, one could argue that a better understanding of the time and (intracellular) place at which the dissociation of the complexes occurs could be crucial to fully explain our observations.

Cell Line, Tumor↗

PubMed2001

Estradiol signaling via sequestrable surface receptors.

Estradiol (E(2))-signaling is widely considered to be exclusively mediated through the transcription-regulating intracellular estrogen receptor (ER) alpha and ERbeta. The aim of this study was to investigate transcription-independent E(2)-signaling in mouse IC-21 macrophages. E(2) and E(2)-BSA induce a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) of Fura-2 loaded IC-21 cells as examined by spectrofluorometry. These changes in [Ca(2+)](i) can be inhibited by pertussis toxin, but not by the ER-blockers tamoxifen and raloxifene. The E(2)-signaling initiated at the plasma membrane is mediated through neither ERalpha nor ERbeta, but rather through a novel G protein-coupled membrane E(2)-receptor as revealed by RT-PCR, flow cytometry, and confocal laser scanning microscopy. A special feature of this E(2)-receptor is its sequestration upon agonist stimulation. Sequestration depends on energy and temperature, and it proceeds through a clathrin- and caveolin-independent pathway.

Animals↗

PubMed1997

Cell surface expression of two major yeast allergens in the Pityrosporum genus.

BACKGROUND: We have previously identified two major allergens of Pityrosporum orbiculare and characterized these as 37 kDa and 67 kDa proteins. OBJECTIVE: In the present study we have investigated the presence and subcellular location of the 37 kDa and 67 kDa allergen components in various members of the genus Pityrosporum as well as in Candida albicans, Candida parapsilosis and Saccharomyces cerevisiae. METHODS: To detect both cell surface and intracellular expression of the allergens, flow cytometry and confocal laser scanning microscopy (CLSM) were used. The cells were stained with indirect immunofluorescent (IIF) or alkaline phosphatase anti-alkaline phosphatase (APAAP) methods using mouse monoclonal antibodies (MoAbs). RESULTS: Ninety-five per cent of the P. orbiculare (P. ovale) cells cultured for 4 days showed cell surface-binding of the anti-37 kDa MoAb and 88% of the cells bound the anti-67 kDa MoAb when analysed with IIF and flow cytometry. It was found that the members of the genus Pityrosporum (Malassezia), P. pachydermatis and M. sympodialis, expressed the 37 kDa and 67 kDa allergens to a similar extent as did P. orbiculare. Less than 5% of the cells of the genus Candida and S. cerevisiae showed positive staining with the MoAbs. The CLSM revealed that the 37 kDa and the 67 kDa components were located to the cell wall and could not be detected inside the acetone fixed and APAAP stained yeast cells of the genus Pityrosporum. When the yeast cells were cultured for more than 4 days the expression of both allergens decreased significantly. CONCLUSION: All three members of the genus Pityrosporum express the 37 kDa and 67 kDa major allergens on the cell surface, whereas these proteins could virtually not be detected in the Candida genus and S. cerevisiae.

Allergens↗

PubMed1997

Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their N-oxides in intact human cells using 647-nm krypton laser excitation.

Flow cytometry and laser-scanning confocal fluorescence microscopy have been used in the study of the pharmacodynamics, in single intact cells, of two novel alkylaminoanthraquinones (AQ4 and AQ6), structurally based upon the mid-red excitable but very weakly fluorescent anticancer agent mitoxantrone, together with their respective N-oxide derivatives (AQ4NO and AQ6NO). The drug design rationale was that N-oxide modifications generates prodrug forms suitable for selective bioreductive-activation in hypoxic tumor cells. DNA-binding ranked in the order of mitoxantrone > AQ6 > AQ4 > AQ6NO >> AQ4NO. Using both cytometric methods a similar ranking was found for whole cell and nuclear location in human transformed fibroblasts. However, AQ6 showed enhanced nuclear uptake compared with mitoxantrone, in keeping with its greater capacity to inhibit DNA synthesis. Partial charge neutralisation by N-oxide derivatization resulted in loss of DNA synthesis inhibition but retention of the ability to accumulate in the cytosol, an important property for prodrug development. We conclude that both flow cytometry and confocal imaging revealed biologically significant differences between analogues for subcellular distribution and retention properties. The study demonstrates the potential for these complementary 647-nm krypton laser line-based fluorometric methods to provide relevant structure-activity information in anthraquinone drug-design programmes.

Anthraquinones↗

PubMed2001

Cytometric analysis of an epitope-tagged transit peptide bound to the chloroplast translocation apparatus.

Chloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope-tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His-S-SStp, contains a removable dual-epitope tag, His-S, at its N-terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser-scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C-terminal to the transit peptide, His-S-SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non-radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre- and post-import thermolysin treatment, photochemical cross-linking and organelle fractionation. The interaction was specific for the transit peptide since His-S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP-dependence. The addition of GTP showed inhibition of the binding of His-S-SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His-S-SStp and Toc75 by confocal microscopy revealed a patch-like labeling, suggesting a co-ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast-binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.

Amino Acid Sequence↗

PubMed2003

Selective internalization of monoclonal antibodies by B-cell chronic lymphocytic leukaemia cells.

B-cell chronic lymphocytic leukaemia (B-CLL) cannot be cured by conventional chemotherapy, therefore, toxin-linked therapeutic monoclonal antibodies (mAbs) are increasingly examined for their potential to improve clinical outcome. The current study aimed to identify mAbs that were internalized by the B-CLL cells of 14 patients, using both flow cytometry and confocal laser scanning microscopy. Anti-CD5, CD22 and CD40 mAbs were effectively taken up by B-CLL cells, whereas mAbs against CD19, CD20, CD23 and CD45 were not. This study may form a basis for further research to identify antibodies that may serve as carriers for toxins to treat B-CLL.

Antibodies, Monoclonal↗

PubMed2005

Effect of all-trans-retinoic acid on mRNA binding protein p62 in human gastric cancer cells.

p62 is a cancer-associated antigen binding to mRNA encoding insulin-like growth factor II that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). In the present study, multiple methods including flow cytometry, confocal laser-scanning microscope, electron microscope were used to characterize the effect of ATRA on BGC-823 cells, which presented two phenotypes of differentiation and apoptosis in cells treated with 1.0 and 50 microM ATRA, respectively. Interestingly, we found that p62 was cytoplasmic in location, but it significantly decreased in cytoplasm and appeared in nucleus of cells when the cells were treated with 50 microM all-trans retinoic acid (ATRA) for 5 days. Furthermore, proteomics approach on differential nucleus proteins showed that the up-regulation and/or down-regulation of cell cycle proteins and IGF binding proteins were involved in the apoptosis of BGC-823 cells induced by ATRA. These results suggest that there is a significant association between expression and distribution of p62 and the growth arrest of tumor cells, in which p62 is associated with cell apoptosis induced by ATRA.

Apoptosis↗

PubMed2002

Uptake of poly(D,L-lactic-co-glycolic acid) microspheres by antigen-presenting cells in vivo.

Dendritic cells are the most potent antigen-presenting cells (APC) and the most effective stimulators of primary T cell responses. Based on the strong influence of the APC on the immune response, we investigated cellular uptake of a biodegradable antigen delivery system, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, at two sites of injection: intraperitoneal and intradermal. We hypothesized that a fluorescent probe, tetramethylrhodamine labeled dextran, loaded in PLGA microspheres would be taken up by APCs and thereby provide a means for studying cellular uptake of PLGA microspheres in vivo. Phagocytic load and cell phenotype were determined using flow cytometry and confocal laser scanning microscopy. The results revealed cellular uptake of tetramethylrhodamine dextran loaded PLGA microspheres at both injection sites. After intraperitoneal immunization, the predominant cell phagocytosing PLGA microspheres in the peritoneal cavity was the macrophage whereas the intradermal immunization resulted in uptake of PLGA microspheres by dendritic cells. Hence, these results suggest that the profile for cellular uptake varies with the site of injection. More importantly, this study provides direct and conclusive evidence of uptake of PLGA microspheres by the most potent APC, the dendritic cell.

Animals↗