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Biotechnological-Biomedical Center (BBZ) BioCity Leipzig

2–4 April 2009

14. LEIPZIGER WORKSHOP

Cytomics and Regenerative Medicine

Incorporating: 7th International Workshop, Slide-Based Cytometry

Organisation:

Prof. Dr. A. Tárnok

Paediatric Cardiology, Heart Centre Leipzig GmbH University Hospital

Prof. Dr. A. Robitzki

Biotechnological-Biomedical Center (BBZ) University of Leipzig

Prof. Dr. U. Sack

Institute of Clinical Immunology and Transfusionmedicine, University of Leipzig

Prof. Dr. F. Emmrich

Institute of Clinical Immunology and Transfusionmedicine, University of Leipzig

LECTURES

Abstract no. L01

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SONATA A TRE: HITTING THE RIGHT TARGET IN HEMATOLOGICAL MALIGNANCIES?

Ghiotto F, Fais F, Parodi S, and Bruno S
University of Genova

Evasion of apoptosis is a central step in tumor development and resistance to cytotoxic therapy. One mode of apoptosis is triggered by intracellular death signals that perturb mitochondria. This so called intrinsic pathway of programmed cell death is regulated by a network of proteins of the B cell lymphoma-2 (Bcl-2) family, which comprises multi-domain anti-apoptotic and pro-apoptotic members, and a large group of pro-apoptotic single-domain members, the BH3-only proteins. The BH3-only members of the Bcl-2 family are critical for cell death initiation and exert their activity by selectively binding either to specific anti-apoptotic members and/or multi-domain pro-apoptotic members. A tightly tuned, although still mostly elusive, interplay among these proteins governs the cell's fate.

Based on the observation that apoptosis resistance in tumor cells is frequently due to overexpression of a pro-survival Bcl-2 member, or to impairment of the activation of pro-apoptotic BH3-only proteins, the features of the latter have been recently exploited for the development of rationally designed therapeutic anticancer agents that should mimic their mode of action thereby restoring the natural process of programmed cell death. Accordingly, several peptidic and non-peptidic BH3 mimetic molecules have been produced in the last decade.

Here we report an overview of the biological properties of the most relevant peptidic BH3 mimetic molecules, in single agent as well as combination settings. Selectivity of their cytotoxic action for cancer cells with respect to normal cells will be discussed. In particular, the apoptogenic and anti-leukemic activity of a novel Bim-BH3 like molecule on in vitro, ex vivo and in vivo hematological tumor cell systems will be described, according to results obtained by cytometric technologies such as multiparameter flow cytometry and confocal microscopy.

Abstract no. L02

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NEW ADVANCES IN MULTIPARAMETER FLOW CYTOMETRY DATA ANALYSIS APPROACHES BY THE EUROFLOW GROUP

Orfao A
Department of Medicine, Cytometry Service and Cancer Research Centre (IBMCC-CSIC/USAL), University of Salamanca

In the last decades, multiparameter flow cytometry immunophenotyping has become an essential technology in cell diagnostics. Accordingly, during this period, its clinical utility has quickly expanded from HIV (classification and monitoring) to other areas, from which the diagnosis, classification and monitoring of different subgroups of haematological malignancies, remains is currently one of the most relevant. Such progressive clinical utility of multiparameter flow cytometry has been paralleled by impressive technological advances. However, despite the significant advances which have occurred in the capability to acquire flow cytometry data about multiple parameters measured in single cells (for millions of events), data analysis is still frequently based on the definition of expert-selected bidimensional plots, where an operator selects the population of interest and interprets its immunophenotype.

Since 2004, the EuroFlow group has been working in the development and implementation of innovation in clinical flow cytometry, with especial emphasis in leukaemia immunophenotyping. Part of the EuroFlow efforts and recent contributions relate to new data analysis approaches and strategies. Basic concepts behind such strategies require: 1) merging of different datafiles corresponding to distinct aliquots of a sample, 2) virtual correction of differences between corresponding cell populations present in different aliquots of the same sample, and 3) calculation of immunopheotypic data measured in one sample aliquot to the corresponding/similar cells measured in the other aliquots of the same sample. This generates single datafiles per sample which contain information about an unlimited number of parameters in huge numbers of individual cells. From the practical point of view, such strategy can then be used to create template datafiles containing reference cases composed of specific populations of cells from: 1) normal and reactive samples, 2) specific disease entities. The combination with automated multivariate analysis, allows for rapid interpretation of the phenotypic results obtained for a given cell population with a rapid and visual classification of individual cells against the reference populations (e.g. normal vs neoplastic or disease A vs disease B). This type of comparisons allows for: 1) quick evaluation of the panels of reagents used, 2) ranking of the added utility (vs redundance) of individual markers in a panel, 3) simulation of the specificity of a given phenotype (combination of markers) to distinguish between two groups of cells with increasing levels of sensitivity (e.g.: testing the sensitivity of that phenotype for further minimal residual disease detection), and; 4) identification of new subgroups of a given disease.

In this presentation the new concepts and approaches in multiparameter flow cytometry data analysis contributed by the EuroFlow group, will be reviewed.

Abstract no. L03

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HIGH-CONTENT IMAGING CYTOMETRY

Shorte S
Institut Pasteur, Imagopole, Paris

Conventional flow cytometry methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information, a limitation prompting a few commercial manufacturers to produce state-of-the-art flow cytometry systems enabling fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells in flow suspension. We have developed novel methodological approaches to this problem that combine micro-fluidic flow, and microelectrode dielectric-field control to manipulate, rotate and immobilize individual cells in suspension during high speed image acquisition. The methods allow unique possibilities for imaging cells in suspension, including multi-modal three-dimensional (3D) tomography. Further, confocal “axial tomography” using micro-rotation imaging greatly enhances three-dimensional optical resolution and contrast compared with conventional methods. Finally, we show that the method can be adapted to allow low throughput cell sorting, and lends itself to automation suggesting its eventual utility for high-content imaging cytometry.

Abstract no. L04

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PRINCIPLES IN HEPATOCYTE TRANSPLANTATION

Stock P
First Department of Medicine, University of Halle-Wittenberg

Hepatocyte transplantation is an alternative to whole liver transplantation, since there is a serious shortage of donors for the treatment of end-stage liver diseases. It is anticipated that healthy donor hepatocytes integrate into the host liver and thus provide permanent functional replacement. Yet, the cellular and molecular mechansims prompting integration are largely unknown. In animal models of liver regeneration by transplanted hepatocytes efficient repopulation by transplanted cells requires a growth advantage over host hepatocytes and a mitotic stimulus provided by the recipient liver. Under those conditions transplanted donor hepatoytes may nearly completely replace host hepatocytes in the recipient liver. It seems not surprising that both the age of the donor hepatocyte and the age of the recipient liver substantially contribute to the rate of repopulation, older livers obviously being less well suited for hepatocyte transplantation. At present, clinical success of the method is still hampered by the limited availability of suitable donor organs for the isolation of hepatocytes of transplantable quality and quantity. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Recent work has identified candidate stem cells both from hepatic or extrahepatic origin to generate hepatocyte-like cells featuring typical hepatocyte functions both in vitro and in vivo after hepatic transplantation. Yet, little is known on the mechanisms of stem cell differentiation to hepatocytes. It will be a major goal in the future to investigate the basic principles of tissue regeneration by hepatocytes derived from stem cells in order to promote cellular integration and functional hepatic repair. It might be worthwhile considering that different underlying liver diseases, which create different cellular and molecular hepatic microenvironments, require hepatocytes to be transplanted that meet these diverse specifications best, thus enabling the transplanted cells to integrate, proliferate and rebuild the damaged host liver.

Abstract no. L05

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ULTRASTRUCTURAL STUDY ON THE OLFACTORY RECEPTOR CELLS OF PSEUDAPOCRYPTES LANCEOLATUS (BLOCH AND SCHNEIDER)

De SK, and Sarkar SK
Electron Microscope and Fish Biology Research Unit, Department of Zoology, Vidyasagar University

The present study deals with the ultrastructural investigation on the olfactory structure of Pseudapocryptes lanceolatus (Bloch and Schneider), a teleostean gobiid fish of gangetic bengal, India, which comprises of single olfactory lamella on either side along with lachrymal and ethmoidal sac. To examine ultrastructure of olfactory receptor cells, olfactory lamella was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2–7.4) for 1hour at 40C under transmission electron microscope (Morgagni - 268D) at 80kV. The olfactory epithelium is a pseudostratified structure. There are two types of olfactory receptor cells have been identified viz., ciliated receptor cells and microvillous receptor cells. The receptor cells are typical bipolar neuron. The apical tip of the receptor bulges to form olfactory knob. The olfactory knob bears cilia in ciliated receptor cells and microvilli in microvillous receptor cells respectively. The number of the cilia varies from 3 to 6. The transverse section of the cilia shows (9 2) arrangement of microtubules. The body of the cilia is implanted in the cytoplasm just beneath the plasma membrane. The basal body is a complex structure which comprises centrioles and associated with numerous neurofilaments as in higher vertebrates. The microvillous receptor cell also possesses neurofilaments in their cytoplasm. Olfactory knob and its associated structures indicate the sensory nature of ciliated and microvillous receptor cells. Chromatinized nucleus present in the lower region of the perikaryon of sensory receptor cells. The number and morphology of the mitochondria in the sensory cell shows specific structural demarcation. Usually huge amount of mitochondria are aggregated at the apical portion of the receptor cell. The subcellular organelles i.e., Golgi apparatus, rough endoplasmic reticulum (rER), lysosomes and free ribosomes are well marked in the cytoplasm of sensory receptor cell. The sensory cell is well adhered with adjace cell by desmosomes. This study reveals that the apical tip of the dendron is a specialized structure because it comes into close contact with the water which is present in the nasal cavity during the water ventilation process through the nostrils over the olfactory epithelium.

Abstract no. L06

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COMBINED HIGH THROUGHPUT AND HIGH CONTENT ANALYSIS OF NON-ORGAN SPECIFIC AUTOANTIBODIES

Großmann K,1 Hiemann R,1 Schröder C,1 Sack U,2 and Conrad K3
1Lausitz University of Applied Sciences 2Institute of Clinical Immunology and Transfusion Medicine University of Leipzig 3Institute of Immunology, Medical Faculty of the Technical University Dresden

Motivation: The detection of autoantibodies for the diagnosis of autoimmune diseases could be faster, more efficient, and more cost effective by combined screening and differentiation. Most laboratories detect autoantibodies with diverse test principals and beyond different test systems. For high quality and perhaps standardized determination of non-organ specific autoantibodies, a cost efficient combination of screening (high throughput analysis) and differentiation of autoantibody specificities (high content analysis) is required.

Methods: For screening with HEp-2 cell array and differentiation with multiplex bead array an inverse fluorescence microscope with image analysis software was used. Beads were coded by ratios of two fluorescent dyes. Third fluorescence measured in every bead population reflected the average antibody reactivity of the sample. Overall 267 sera of healthy blood donors and 84 defined serum samples of patients with SLE or systemic sclerosis which were positive for La/SS-B, CENP-B, Ro60, Ro52, Scl-70, and RNP/Sm were screened on HEp-2-cells. Thirty nine on HEp-2 cell array preclassified negative serum samples of healthy blood donors were randomly selected as negative controls and 84 defined positive serum samples were selected for differentiation with the multiplex bead array.

Results: A total of 123 serum samples were compared to determine the concordance of the multiplex bead array with commercial available enzyme immunoassays (EIA) for the reaction against five defined autoantigens. The results for autoantibody positive sera obtained by the multiplex bead array fit well with the results of the EIAs. CENP-B, Ro60, and Ro52 had a greater than 93 % relative sensitivity and about 98 % relative specificity.

Conclusion: The system allows combined high content and high throughput analysis of non-organ specific autoantibodies. Thus, the screening as well as the differentiation of autoantibodies can be performed on the same technological platform.

Abstract no. L07

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RED BLOOD CELLS AGEING, CHARACTERIZED BY, VECTOR CONTRAST CONFOCAL ACOUSTIC SCANNING MICROSCOPY

Ahmed Mohamed E,1 Zschörnig O,2 Willekens F,3 Kamanyi A,1 Von Buttlar M,1 Wannemacher R,1 and Grill W1
1Institute of Experimental Physics II, University of Leipzig, Linnéstr. 5 2Institute of Medical Physics and Biophysics, Härtelstr. 16-18, 04107 Leipzig 3Department of Clinical Chemistry, Rijnstate Hospital, PO Box 9555, 6800 TA, Arnhem, The Netherlands

Background: The contrast in acoustic microscopy depends on the mechanical properties of the object. Red blood cells are known to be highly elastic cells capable of modifying their biconcave shape to adapt to narrow passages in areas of microcirculation. During aging the erythrocytes increase their rigidity until they reach the senescent state and are removed. The process of recognition and removal of senescent cells is still under discussion and might also be influenced by alterations in the elastic properties leading to a reduced deformability.

Material and Methods: Red blood cells were fractionated into different age groups, smeared on glass slides, fixated with methanol and imaged in three dimensional scans.

The smears were Imaged with vector (phase and magnitude) contrast confocal scanning acoustic microscopy in reflection operated at 1.2 GHz with a lateral resolution of 1 μm.

Results: The data is analyzed concerning correlations of the age of the cells and their appearance in phase and magnitude contrast.

Discussion and conclusions: The results are presented and discussed.

Abstract no. L08

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IN VIVO GENERATED TH17 CELLS HAVE A STABLE MEMORY FOR IL-17 EXPRESSION

Lexberg M,1 Foerster A,2 Richter A,2 Radbruch A,1 and Chang HD1
1DRFZ Berlin 2Miltenyi Biotec, Bergisch Gladbach

T helper (Th) lymphocytes regulate immune responses through the secretion of cytokines. Which cytokines they secrete is determined by costimulatory signals they receive during their primary activation by antigen. Based on the expression of cytokines, Th cells have been classically divided into Th1 and Th2 cells. Recently a novel lineage of CD4+ T cells has been added which is characterized by the production of IL-17. The memory expression of cytokines for Th1 and Th2 has been well studied and has been shown to depend on the imprinting of cytokine genes and the differential expression of master transcription factors such as T-bet for Th1 cells and GATA-3 for Th2 cells. We have developed a cytometric IL-17 secretion assay for the isolation of Th cells based on their expression of IL-17. It was our aim to analyse the stability and plasticity of IL-17 expression in Th17 cells on the single cell level. When isolating IL-17 expressing cells from Th17 cells generated in vitro according to state-of-the-art protocols in the presence of TGF-ß, IL-6, IL-23, anti-IL4 and anti-IFNγ, we observed that these cells do not reexpress IL-17 upon in vitro stimulation in the absence of the original instructive signals. In response to IL-12 or IL-4 in vitro generated Th17 cells are converted to Th1 or Th2 cells, respectively. In contrast, IL-17 expressing Th cells isolated directly ex vivo maintained IL-17 expression upon in vitro culture and were refractory to Th1 or Th2 polarization. Apparently, Th17 cells are stably imprinted for IL-17 expression in vivo by signals not provided in protocols for the in vitro generation of Th17 cells. Our results show that Th17 cells generated in vivo are indeed a stable Th cell lineage distinct from Th1 and Th2 cells and functionally imprinted for IL-17 expression. We are currently studying the molecular basis for the stability of IL-17 expression in vivo.

Abstract no. L09

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DEFINITION OF LEUKOCYTE SUBSETS IN PRIMATE BRAIN BY POLYCHROMATIC FLOW CYTOMETRY

Bischoff T, Baumgart A, Mätz-Rensing K, Stahl-Henning C, and Sopper S
Deutsches Primatenzentrum

Macaques are commonly used for studying neurological disorders, including MS and HIV-1 associated dementia. Flow cytometric characterization of the intrathecal immune response in these diseases was restricted due to the paucity of the material, the heterogeneous composition of the cells and the high autofluorescence of microglia cells.

We have used multicolor flow cytometry to define leukocyte subsets in cell suspensions isolated from enzymatically digested macaque brain tissue by density gradient purification.

Based on a systematic approach, we established a combination of antibodies directed against 9 different antigens (CD3, CD8, CD11b, CD11c, CD14, CD16, CD20, CD45, and CD159a) allowing the definition of microglia, monocytes/macrophages, dendritic cells, B-cells, NK-cells and T-cells in a single tube. In addition, this antibody combination also enables the discrimination of functionally different subsets among the leukocyte populations such as CD16 positive “inflammatory” macrophages. Using this antibody cocktail a more accurate determination of microglia, usually defined only by a distinct expression of CD45 and CD11b, could be achieved based on the absence of markers specific for other subsets such as CD14 and CD11c. At a proportion of about 95%, microglia cells are the major leukocyte subset in the brain. Two other myeloid cell populations, CD14 macrophages and CD11c dendritic cells were also identified. In contrast to blood, where macrophages are more abundant, dendritic cells outnumbered macrophages in the brain. Among lymphocytes, CD20 positive B-cells were decreased and T-cells as well as NK-cells were increased in the brain compared to blood.

Our data showed a non-exclusive expression of certain surface makers on different cell populations. Therefore it is necessary to use polychromatic flow cytometry, defining at least nine antigens in one reaction, for a correct classification of major leukocyte subsets in the brain.

Abstract no. L10

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FLOW CYTOMETRY AS A POTENTIAL INSTRUMENT FOR NON-INVASIVE SIMPLE METHOD OF MILD ENDOMETRIOSIS DIAGNOSIS

Chrobak A,1 Szopa M,1 Rossowska J,2 Sobczynski M,1 and Gabrys M3
1Faculty of Biotechnology, University of Wroclaw, Poland, 2Institute of Immunology and Experimental Therapy Polish Academy of Science, Wroclaw, Poland 3First Department of Gynaecology and Obstetrics, Medical University in Wroclaw, Poland

Background: Endometriosis is a gynaecological disease which affects up to 15% of all women in reproductive age. It causes pain, abnormal bleeding and infertility. Its aetiology is unknown but immunological disturbances are well documented. Up to now the only certain way to diagnose endometriosis is to make a laparoscopy. Efforts of many laboratories head towards finding a simple, fast and non-invasive method for diagnosing this disease. We decided to study the differences in percentage of subpopulations of blood T cells in infertile women with endometriosis in comparison to healthy ones or with unexplained infertility.

Material and methods: Pilot studies were done. Peripheral blood mononuclear cells were isolated from forty women at reproductive age: 22 with endometriosis, 11 with simple ovarian cysts (control group) and 7 with unexplained infertility. No patient had been treated with hormonal therapy minimum three months before the studies. Cells were analysed by four-coloured flow cytometry with using monoclonal antibodies anti-CD4, CD8, CD29, CD95, FasL, CD69, CD25, CD34. There were analysed one, two, three and four coloured populations in WinMDI 2.9 program. There were bootstrap MANOVA, next bootstrap ANOVA and Tukey HSD tests used for statistic analyzing of data.

Results: We have shown that there were significant differences in percentage of cell populations: CD8 CD29 CD69, CD8 CD29 CD69 CD95, CD8 CD29 FasL and CD8 CD95 between women with mild endometriosis and control fertile patient. Additionally, a logistic model for mild endometriosis was performed.

Conclusions: Immunophenotyping by flow cytometry seems to be a promising method for diagnosing the mild endometriosis.

Abstract no. L11

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IMPACT OF ß-CATENIN ON HEPATOCYTE DIFFERENTIATION OF HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS (MSC)

Sgodda M and Christ B
University of Halle-Wittenberg

Background: The molecular processes underlying hepatocyte differentiation of MSC are not known. There is some indication that ß-catenin signaling might be involved.

Materials and methods: MSC were selected from human peritoneal adipose tissue (phAT-MSC) by plastic adherence, differentiated in specified medium and molecular components of ß-catenin signaling studied in vitro by immunocytochemistry, Western blot and analysis of transgenic expression of luciferase under the control of a ß-catenin target promotor.

Results: Hepatocyte differentiation of phAT-MSC resulted in the translocation of ß-catenin from the cytoplasm to the nucleus. The amount of phosphorylated ß-catenin decreased, and in the presence of the GSK3-ß inhibitor BIO, ß-catenin was found also in the nuclei of undifferentiated phAT-MSC consistent with stabilisation and nuclear translocation of ß-catenin during differentiation. In transgenic cells, luciferase activity was high in undifferentiated cells and decreased during hepatocyte differentiation. ß-catenin target genes were hardly expressed in undifferentiated phAT-MSC but increased with hepatocyte differentiation. In the presence of BIO, proteins were also expressed in undifferentiated cells indicating a ß-catenin-mediated response. In the liver, the expression of APC increases from perivenous to periportal hepatocytes triggering expression of marker genes like PCK1 in periportal and of GS in perivenous hepatocytes. GS, APC and PCK1 were not expressed in undifferentiated phAT-MSC but APC and PCK1 expression increased with hepatocyte differentiation. In the presence of BIO, GS was expressed instead.

Conclusions: The decrease of ß-catenin mediated gene activation during hepatocyte differentiation of phAT-MSC might indicate down-regulation of ß-catenin-dependent gene activation. Yet, nuclear translocation points to a pivotal role of ß-catenin in the determination of a periportal or perivenous hepatocyte phenotype during differentiation.

Abstract no. L12

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FROM TRANSCRIPTOME TO CYTOME: PUTTING CANDIDATE GENES IN A GLOBAL IMMUNOPHENOTYPICAL CONTEXT

Gruetzkau A,1 Steinbrich-Zoellner M,2 Gruen J,1 Kaiser T,1 and Radbruch A1
1Deutsches Rheuma-Forschungszentrum Berlin 2Rheumatologie, Charité (CBF)

Background: Gene expression studies of peripheral blood cells in inflammatory diseases revealed a large array of new antigens as potential biomarkers useful for diagnosis, prognosis and therapy stratification. State-of-the-art multicolour flow cytometry makes it attractive to validate candidate genes accompanied by a detailed immunophenotyping of blood cell subsets.

Aim: A translational approach should be established, that would help to validate disease-specific candidate genes at the protein and single cell level by multiparametric flow cytometry. In addition, this approach should integrate the possibility for a complex immunophenotyping of peripheral blood or bone marrow samples to get information about the disease state responsible for the expression of a particular candidate gene.

Methods: We developed a set of multicolour staining panels that allowed the assessment of several hundreds of phenotypical parameters in a few milliliters of peripheral blood. Up to ten different surface antigens were measured simultaneously by the combination of seven fluorochromes. In a pilot study, blood samples of ankylosing spondylitis (AS) patients were compared to normal donors (ND) with respect to identify immunophenotypic signatures useful for disease classification.

Results: We could establish a set of multicolour stainings that allowed monitoring of all major leukocyte populations and their corresponding subtypes in peripheral blood. The feasibility of our cytometric profiling approach was demonstrated by a successful classification of AS samples by a limited set of phenotypical parameters. These parameters allowed an error-free prediction of independent AS and ND samples originally not included for parameter selection.

Conclusion: Altogether, this study demonstrates a new level of multiparametric analysis of candidate genes at the single cell level in the post-transcriptomic era that will be helpful with respect to the requirements of a personalized medicine approach.

Abstract no. L13

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VERIFICATION OF GOLD NANOPARTICLE UPTAKE BY BOVINE IMMORTALISED CELLS USING LASER SCANNING CONFOCAL MICROSCOPY

Taylor U,1 Petersen S,2 Barcikowski S,2 Rath D,1 and Klein S1
1Institute of Farm Animal Genetics, Friedrich Loeffler Institute, Federal Research Institute of Animal Health 2Laser Zentrum Hannover e.V.

Due to their surface plasmon resonance, gold nanoparticles (GNP) possess exceptional optical properties. Furthermore if laser-generated, they can be conjugated to ligands such as oligonucleotides and proteins with particular ease and are exceptionally stable in solution without the need of additives. They thus appear to be ideal biomarkers. However, to actually visualize them remains a challenge, which is mainly due to the nanoparticle specific complex interactions between light absorption, extinction and scattering. The present work was designed to study the cellular uptake of laser-generated GNPs into a bovine cell line using a laser scanning confocal microscope (LSM 510; Zeiss). Acquisition standards were set up using the GNP-solution as positive and untreated cells as negative controls. Cultured bovine immortalised cells (GM 7373) were coincubated with GNPs (average diameter 15 nm, 10 μg/ml) for 2, 24 and 48 h. The light scattering caused by the intracellular GNPs was detected by applying the multitracking mode. This set-up facilitated a clear distinction between GNP-containing coincubated cells and the negative controls. It also confirmed that the GNPs were positioned intracellular and not membrane-associated. The cells incorporated the GNPs in a time dependent manner. In conclusion, cellular uptake of GNPs takes place during coincubation and can be visualized using laser scanning confocal microscopy.

Abstract no. L14

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TRIPLE FUNCTIONALISED CACO3 PARTICLES AS COMBINED SENSOR AND TRANSPORT SYSTEM FOR ACTIVE AGENTS IN CELLS

Reibetanz U,1 Chen MH,1 Liaw ZY,1 Oh HL,1 Donath E,2 and Neu B1
1Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 2Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Germany

In recent years colloidal particles and capsules in sub-micrometer dimensions, Layer-by-Layer (LbL) coated with biocompatible poly-electrolytes, have received much attention as drug delivery systems. In order to establish such particles as transport systems it is not only necessary to get a detailed understanding of the fabrication and functionalisation of these capsules but also of the processing within the cell including the release of the particle from the endo-lysosome into the cytoplasm and eventually of the active agent. In this study we successfully functionalized LbL-coated micro-particles as combined sensor and transport systems in order to study the time dependent release of an active agent into cells.

CaCO3 particles were functionalised with fluorescein isothiocyanate (FITC-PAH), a pH sensitive fluorescent dye, to allow particle localisation in the endo-lysosome and cytoplasm. The labelled core was than coated with consecutive layers of a Rhodamine-B-Isothiocyanate labelled protamine (RITC-PRM) and dextran sulphate (DXS). Plasmid DNA (pEGFP-C1) as a reporter was integrated into the biocompatible and biodegradable PRM/DXS multi-layer. The processing of the active agent, represented by the reporter plasmid DNA, the multi-layer degradation after RITC-PRM release and the localisation of these particles were than investigated in HEK293T cells via flow cytometry and confocal laser scanning microscopy. Our results show that the combination of a sensor and transport function in the same system allows the quantitative study of the processing of such carriers in cells and should thus be a useful tool to study the efficiency and time-dependence of the release of active agents into cells.

Abstract no. L15

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EVALUATION OF PERFORMANCE AND APPLICATIONS OF LUMISENS DIGITAL IMAGING SYSTEM

Pierzchalski A,1 Müller HW,2 Bocsi J,3 and Tarnok A3
1Translational Center for Regenerative Medicine, University of Leipzig 2Sensovation AG, Stockach, Germany 3Department of Pediatric Cardiology, Heart Center Leipzig, University Leipzig, Germany

Microscope imaging systems become the standard in research and clinical diagnostics. The key aspects for their robust application are automatization and data processing.

The innovative LumiSens (Sensovation AG) slide-based technology based on fluorescence digital imaging microscopy was used. The instrument is equipped with an inverted microscope, blue and red LEDs, double band pass filters and high-resolution cooled 16-bit digital camera. The instrument was evaluated for its performance compared with other flow and slide-based cytometers. The dynamic range for rainbow beads (Spherotec Inc.) measurement was estimated revealing high sensitivity and similar resolution as for other standard methods. The instrument revealed the ability to detect up to 7 bead populations showing its high-performance.

LumiSens proved to be useful for absolute cell counting of leukocytes in peripheral blood which gave results comparable to routine laboratory counting. The instrument was also evaluated for its capability for immunophenotyping of CD4 or CD22 positive events. Using To-Pro3 or SytoxGreen DNA dyes the cells were identified and CD4 or CD22 positive events were counted giving the similar results to those obtained by flow cytometry.

The sensitivity of the equipment was moreover confirmed by evaluation of the CD3 antigen expression on CD4 and CD8 positive cells which gave expected results confirmed by flow cytometry measurements.

Using CD45 and CD14 staining it was also possible to obtain differential peripheral blood leukocyte picture which gave the comparable results to flow cytometry and routine laboratory values.

Finally the sensitivity of LumiSens apparatus was verified by estimation of DNA cell cycle distribution on C2C12 human skeletal muscle cell line. The cells were easily identified and the phases of the cell cycle could be shown.

LumiSens digital imaging system is a promising, versatile and cost-effective alternative for other slide-based cytometers present in the market.

Abstract no. L16

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UP-REGULATION OF CD40-CD40 LIGAND SYSTEM ON PLATELETS IN PATIENTS WITH SEPSIS - FLOW CYTOMETRIC EVALUATION OF PLATELET ACTIVATION

Wolf Z,1 Ugocsai P,1 Wittmann S,2 Luchner A,3 Müller T,3 Wrede C,4 Spanuth E,5 and Schmitz G1
1Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany 2Department of Anaesthesiology, University of Regensburg, Regensburg, Germany 3Department of Internal Medicine II, University of Regensburg, Regensburg, Germany 4Department of Internal Medicine I, University of Regensburg, Regensburg, Germany 5Dianeering GmbH, Diagnostic Engineering, Research and Know-How Services, University technology Park, Heidelberg, Germany

Background: Haemostatic abnormalities and thrombocytopenia are common in critically ill patients and correlate with the severity of disease. Recent evidence indicates that CD40-CD40 ligand pathway may play a key role in the inflammatory response of platelets. Therefore, our aim was to investigate the effect of sepsis on platelet activation, especially on the CD40-CD40L system, as well as on a- and dense-granule release.

Material and methods; Thirty-nine patients hospitalized with sepsis and 18 healthy controls were evaluated. Platelet surface expression of CD40, CD40L, a-granule marker CD62P, dense-granule protein CD63 and PAC1 (which recognizes only the conformationally activated GPIIb/IIIa) were quantified by flow cytometry ex vivo and after in vitro agonist-induced activation with ADP and TRAP-6.

Results: Patients with sepsis showed significantly enhanced surface expression of CD40, CD40L, CD62P, CD63 and PAC1 compared to controls (p<0.05). a- and dense-granule secretion and PAC1 binding to GPIIb/IIIa remained preserved in septic patients. However, an altered release of CD40L was observed after agonist-induced activation. CD62P, indicator of platelet activation, showed a positive correlation (r>0.5, p<0.002) with surface expression of CD40L, CD40, CD63 and PAC1. Moreover, an inverse correlation (r<−0.45, p<0.001) was observed between these receptors and platelet counts, a predictive marker of a poor clinical outcome. The severity of sepsis defined by SAPS score also correlated (r>0.37, p<0.05) with the activation state of platelets.

Discussion and conclusions: Our data demonstrate that sepsis is associated with a higher activation state of platelets, particularly with an up-regulation of the CD40-CD40L system. Sepsis does not affect the secretion of a- and dense-granules, however, alters CD40L production of a-granules. These findings suggest that the augmentation of the CD40-CD40L system participates in the amplification of platelet inflammatory response in sepsis.

Abstract no. L17

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A NEW FLOW CYTOMETRY BASED SYSTEM TO DETECT AND QUANTIFY HIV-1 IN BLOOD SAMPLES

Weidner J,1 Greve B,2 Van Dülmen A,2 Cassens U,3 Sibrowski W,4 Nasdala I,1 and Göhde W5
1Partec GmbH, Am Flughafen 13, 02828 Görlitz, Germany 2Department of Radiotherapy-Radiooncology, University Hospital Münster, 48149 Münster, Germany 3Institute of Transfusion Medicine, Laboratory Medicine and Medical Microbiology, Klinikum Dortmund gGmbH, 44137 Dortmund4Department of Transfusion Medicine, University Hospital Münster, 48149 Münster, Germany 5Medical Faculty, University Hospital Münster, 48149 Münster, Germany

The treatment of HIV-1 infection by highly active antiretroviral therapy (HAART) is monitored preferentially by two different blood parameters, the CD4 T-cell count or CD4% value for children and the plasma HIV-1 virus concentration. The CD4 T-cells decrease during infection and they are monitored by flow cytometry which has been declared as gold standard by the WHO. The HIV copies should remain as low as possible to minimize mutations and is recorded by PCR-based viral load tests. Both parameters are decision supports for the initiation of HAART and used to assess treatment success.

Most commercially available viral load assays are optimised for subtype B, predominant in the western world, but 95% of the infected individuals reside in countries where other subtypes exist. Furthermore, these tests are very expensive and require special equipment.

We explored an alternative technical solution and combine flow cytometry technique with a new PCR test system to detect and quantify all currently known HIV-1 subtypes.

A three primer system was used to amplify an internal control and the clinical probe. The forward primers were coupled either to Dinitrophenole (DNP) or Digoxygenin (DIG) to allow the PCR products to be captured with different sized microbeads coated with Anti-DNP or Anti-DIG antibodies. A Biotin label of the reverse primer enabled the binding of Streptavidin-R-Phycoerithrin to the PCR-fragment and fluorescence detection by flow cytometry. The mean fluorescence intensity correlates over a wide range with the HIV-1virus concentration per ml blood.

This versatile technology has already been adapted to detect proviral HIV-1 DNA which is important e.g. for mother to child virus transmission. One future aspect will be the measurement of HIV-2 viral load and HIV-2 proviral DNA.

The goal is to measure all HIV/AIDS therapy relevant parameters with one device to enable optimal and extremely cost-effective therapy monitoring to improve HAART in the developing world.

Abstract no. L18

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PUBLICATION IN CYTOMETRY PART A, MIFLOWCYT AND DATA PRESENTATION

Tárnok A
Dept. of Pediatric Cardiology, Cardiac Center Leipzig GmbH, University of Leipzig

Publication: Scientific journals require certain minimal standards from a manuscript in order to find them acceptable for further reviewing and publication. There are some very common reasons why a paper does get reviewed and accepted or does not. This tutorial aims to highlight all major aspects of manuscript writing, submission and communication with the reviewers. What can and very often does go wrong and how to do it right in order to improve your chances to get your paper published. Special emphasis will be taken to highlight special needs for a biomedical and technical oriented journal such as Cytometry Part A.

Data presentation: Data from Flow Cytometry (FCM) experiments are frequently presented in a way that they appear incorrect or transport the wrong information or are inconsistent. The most common mistakes and errors in data presentation will be presented. The aim is to help authors to avoid such mistakes in future and help to improve their data presentation quality for presentation and publications. (Based on a tutorial at ISAC 2008, Budapest, Hungary, by Prof. M Roederer, NIH Bethesda).

MIFlowCyt: A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard is presented, stating the minimum information required to report FCM experiments. MIFlowCyt includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. In near future, Cytometry Part A will require for manuscripts with FCM data to follow the MIFlowCyt guidelines. (Cytometry A 2008;73:875, Nat Biotechnol 2008; 26:889).

Abstract no. L19

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SCREENING OF POTENTIAL TOLUENE DEGRADERS IN COMMUNITIES USING A NOVEL FLUORESCENT TOLUENE ANALOGUE AND FLOW CYTOMETRY

Sträuber H, Hübschmann T, Harms H, and Müller S
Hemholtz Centre for Environmental Research-UFZ

Background/Aim: The fast identification and quantification of microbial key players in degradation processes of environmental pollutants will enable for both development of new remediation strategies and understanding of community functioning. A screening method for bacterial toluene-degraders was developed using a new fluorescence dye (NBD-toluene) and flow cytometry.

Material and methods: The staining characteristics of degraders and non-degraders were demonstrated. A fluorescently labelled toluene analogue (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT) and flow cytometry was used to develop an assay for the detection of live individual bacterial toluene-degraders.

Results: Excitation and emission spectra of the new dye were presented showing two absorption peaks (444 nm and 475 nm) and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as the non-toluene-degraders P. putida KT2440 and E. coli K12 were investigated to ensure specificity of NBDT action. NBDT is a membrane permeable dye which is, however, pumped out of the cells by involvement of proton motive force dependent export proteins by live cells. Therefore, CCCP was added to inactivate the export systems and to enable for quantitative analysis of the analogues' uptake into live bacterial cells. NBDT mainly accumulated in the outer membrane of the cells because of its hydrophobic characteristics. Additionally, the dye's channelling by specific toluene transporting porins was assumed because the porin inhibitor cadaverine had a significant influence on the cells' NBDT fluorescence intensity. The latter was found only with the toluene degrading strains P. putida mt-2 and F1 showing 25% and 42% less fluorescence intensity, respectively, in this case. These findings enabled for a differentiation of toluene- and non-toluene-degraders.

Abstract no. L20

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MANUAL CELL COUNTING AND LEUKOCYTE DIFFERENTIATING IN CEREBROSPINAL FLUID CONTROLS OF JOINT GERMAN SOCIETY FOR CLINICAL CHEMISTRY AND LABORATORY MEDICINE (DGKL)

Kleine TO,1 Nebe CT,2 Löwer C,1 Lehmitz R,3 Kruse R,4 Geilenkeuser WJ,4 and Dorn-Beineke A2
1Universitätsklinikum Giessen/Marburg, Abteilung Klinische Chemie und Molekulare Diagnostik2Institut für Klinische Chemie, Universität Heidelberg, 3Klinik für Neurologie & Poliklinik, Universität Rostock, 4DGKL, Referenzinstitut für Bioanalytik Bonn

Background: Cell examination in cerebrospinal fluid (CSF) represents one basic parameter of CSF diagnosis. To evaluate manual cell counting and differentiating, external DGKL trials are carried out with DGKL CSF controls; here data of 10 trials 2004–2008 were evaluated.

Methods: CSF controls with living blood leukocytes and erythrocytes, CSF like, makes the trials feasible to number native leukocytes and erythrocytes microscopically in calibrated chambers (Fuchs-Rosenthal, Neubauer) and to differentiate them visually into mononuclear cells (MNC) and polynuclear cells (PNC) using 7 vital stains: fuchsin in alkaline or glacial acetic acid (Samson); methylene blue stain in aqueous (Bauer) or alkaline solution (Löffler); methylviolet/saponine in glacial acetic acid (Neidhardt); gentian violet in glacial acetic acid (Türk); alkaline toluidine blue. As references, Fuchs-Rosenthal chamber cell counting and immune-flow cytometry (FACScan CD45, CD14) dual platform analysis were applied. Statistical evaluation was done with Passing/Bablock P/B regression analysis.

Results: With respect to references, evaluation of DGKL CSF controls revealed significantly different P/B regressions with lower median counts of leukocytes (50-85%) and erythrocytes (36-75%) as well different MNC and PNC M/L values after vital staining, besides counting and differentiating mistakes.

Conclusions: Evaluation of manual cell counting and differentiating in native DGKL CSF controls revealed inaccuracies with stained cells, caused by cell destructions with acetic (soapy) or alkaline solvents (applied to enter vital stains intracellular); possible exception: erythrocyte staining with Löffler's stain. For CSF diagnosis, native unstained CSF cells are recommended for chamber counting by experienced technologists; it is inaccurate for leukocyte differentiating.

Abstract no. L21

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INNATE VERSUS ADAPTIVE IMMUNE RESPONSE IN PREGNANT WOMEN AND CORRESPONDING CORD BLOODS

Herberth G,1 Hinz D,1 Röder S,2 Diez U,3 Borte M,4 and Lehmann I1
1UFZ-Helmholtz Centre for Environmental Research, Environmental Immunology, Leipzig 2UFZ-Helmholtz Centre for Environmental Research, Core Facility Studies, Leipzig 3Children's Hospital, Municipal Hospital “St Georg”, Leipzig4Children's Hospital, Municipal Hospital “St Georg” and University of Leipzig

Introduction: There is strong evidence that immune responses from newborns differ in comparison to adults. So far, no mother-child data are available. The aim of our study was to analyse the capacity of cord blood cells to respond to innate and adaptive stimulation in terms of Th1/Th2 cytokines and inflammatory markers in comparison to blood cells from the corresponding pregnant women.

Methods: Within the mother-child study LINA (Lifestyle and Environmental factors and their Influence on Newborns Allergy risk) we analysed blood samples from pregnant women in the 34th week of gestation and the corresponding cord bloods for cytokine concentration (n=461 mother-child pairs). Blood samples were left unstimulated, or stimulated with phytohemagglutinin (PHA) or lipopolysaccharid (LPS) for 4 hours. Cytokine concentrations (IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IFN-γ, TNF-α, MCP-1) were measured in the supernatants via flow cytometry using the cytometric bead array (BD Biosciences).

Results: In unstimulated as well as in stimulated samples the inflammatory cytokines IL-6, IL-8, IL-10 and MCP-1 were highly increased in cord bloods whereas IFN-γ and TNF-α concentrations were significantly reduced compared to mothers. In contrast, the Th2 cytokines IL-4, IL-5 and IL-13 were elevated in cord bloods whereas in the stimulated samples these cytokines were reduced compared to mothers. Furthermore, maternal IL-6, IL-8 and MCP-1 concentrations correlated with cord blood levels whereas Th1/Th2 cytokines did not.

Conclusions: Innate immune response via inflammatory cytokines at bird is stronger compared to adults whereas adaptive immune response is strongly reduced.

Abstract no. L22

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FROM SLIDES TO CHIPS - ADVANCING IMAGE CYTOMETRY

Hennig C,1 Adams N,2 and Hansen G1
1Hannover Medical School 2Unilever Centre of Molecular Informatics, Univ. of Cambridge

Background: Cells as building blocks of life are extremely heterogeneous in regard to expressed markersets, activation states or produced compounds. Thus, functional and phenotypic analysis of cells warrants new technological approaches for enabling comprehensive cytometric experiments.

Methods: We developed a technology platform for explorative cytometry that consists of microfluidic chips with cytoadhesive surfaces, a software for microscope-based, iterative cytometry and novell bioinformatic approaches for data-mining and annotation of data.

Results: Validation of our platform against flow cytometry revealed equal specificity but more than 10fold enhanced sensitivity. Living cells of any tested origin like stem cells, immune cells or tumor cells selfimmobilized within the chips. Multiple functional test like calcium flux, receptor shedding and upregulation of activation-associated molecules can be performed, followed by an unlimited intracellular and surface-phenotyping using fluorescent-labelled antibodies. After image recognition, hierarchical cluster analysis of the results turned out to be a useful tool to handle the enormous complexity of cellular data generated when analysing 10000s of cells with a comprehensive markerset of more than 30 markers. However, visualization of the results and semantic-web based annotation of the experimental data are further necessary tools for accessing the complex data produced by this high-content cytometric method. We therefore also present our current bioinformatic solutions in regard to this.

Discussion: In our hands, advancing from slide- to chip-based cytometry combined with appropriate bioinformatics yields several benefits for cytometric experiments thus significantly shortening knowledge generation times. Therefore, we conclude that chip-based cytometry is a useful addition to the cytometrists toolbox.

Abstract no. L23

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QUANTIFYING CELL ADHESION AND CELL STIFFNESS USING SINGLE CELL FORCE SPECTROSCOPY

Mueller T,1 Neumann T,1 and Owen R2
1JPK Instruments AG 2JPK Insttruments AG

The interaction of cells with their external environment plays a key role in multiple cellular processes, from tissue development and cohesion, to cell motility, cancer development and metastasis, and immunology. Using single cell force spectroscopy (SCFC), cell adhesion can be quantified (1), and the contribution of different components e.g. from the extra cellular matrix, can be assessed (2).

Using the atomic force microscope the nano-indentation technique has emerged as a useful tool to determine elastic properties like the Young's modulus for biological samples. Nanomechanical analysis of cells increasingly gains in importance in different field in cell biology like cancer (3), developmental biology (4), and biomaterial research.

We have developed an instrument and workflow to run such measurements on living cells, in a temperature controlled environment in combination with light and fluorescence microscopy. The CellHesion system has two different pulling ranges of 15μm/100μm to investigate the binding forces between cell and target surface, between two cells or between cell and cellular monolayer down to the single-molecule level.

We present a strategy to carry out cell adhesion and cell elasticity experiments using SCFS. The related working parameters for force measurements (e.g. contact time, contact force, and pulling length) and for elasticity measurements (tip geometry, indentation depth, contact point) will be discussed as well as the boundary conditions of the Hertz model (see table).

Abstract no. L24

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TUTORIAL: LASER SCANNING CYTOMETRY

Mittag A
Translational Centre for Regenerative Medicine (TRM Leipzig), University Leipzig, Leipzig, Germany

While flow cytometry is widely known and used in cellular experiments, the technology of Laser Scanning Cytometry (LSC) and its capabilities is still not well-known. Hence, this tutorial should give an introduction in LSC, i.e. the basic principle, data acquisition and analyses as well as some applications. With this technology not only tissue sections and cell cultures can be analyzed but also cells (or other particles) in suspension. Hence, LSC should find its way into common laboratory practice. Although the core component of LSC is a microscope it should not be mixed up with common fluorescence microscopy. LSC yield quantitative data based on fluorescence analyses; comparable to flow cytometry.

Abstract no. L25

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LINK FOR CELL DATA GENERATED BY FLOW CYTOMETRY WITH ASSOCIATED CELL IMAGES

Malkusch W
Carl Zeiss ISG

Flow cytometers quickly provide the data required for large quantity of cells and are able to isolate cell groups. However the direct relationship between the raw data or the parameters and the individual cell image is usually lacking. Isolated cells can no longer be called up and visualized. The AxioVision SFM system was designed to fill this gap. It is by no means intended to replace flow cytometry, but rather to offer the additional benefit of assigning the data to the cell images.

The cells are acquired from cytospin samples in several fluorescence channels using a digital camera via an upright light microscope. They are then measured automatically on the basis of the individual requirements with the help of the image analysis software. The results are combined in a table and correspond to those of a flow cytometer. In addition to densitometric and geometric data, this also contains the cell coordinates of the fluorescence channels and the window coordinates for each individual cell.

Individual cell groups can now be isolated with the help of distributions, for which you define the sequence, number and parameters. The results are displayed for example in the form of a histogram. Now regions of interest may be marked for further distributions of all cells lying within these markings. All values in all measurement channels may be used to create scatter plots. With the help of geometric frames ‘rare events’ may be filtered out. These rare cells are presented in a gallery that also allows for further selection to create another data table.The interesting individual cells can be re-positioned using the object coordinates. This can be done even with a higher magnification.

Abstract no. L26

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AN INTRODUCTION TO FLOW CYTOMETRY – BASIC TUTORIAL

Bocsi J
Heart Center, University of Leipzig, Pediatric Cardiology

Cytometric analyses are frequently used in the clinical diagnostics and biological research. This tutorial will give a brief overview of the principle of cytometry: from the instrument (light sources, emission filters and detectors) to the obtained quantitative data. A plethora of fluorescent dyes for cellular and subcellular staining are available. Therefore, only some characteristics (absorption and emission spectra, stability and quantum efficiency) of the most frequently used fluorescent dyes will be presented. Some practical examples will illustrate the broad application capability of this methodology and its importance in cellular analyses nowadays.

Abstract no. L27

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POLYCHROMATIC CYTOMETRY – PREANALYTICS AND QUALITY CONTROL

Pierzchalski A
Translational Center for Regenerative Medicine, University of Leipzig

Cytometry is a powerful method giving possibility to raise a vast amount of quantitative, valuable data. However cytometric instruments are very complex consisting of many electronic parts. One has to bear in mind that each wrongly designed and not controlled measurement may lead to biased and incorrect result.

Thus, one should follow some general rules which are the prerequisite for obtaining correct and reliable results. Before start one should focus on proper experiment design which comprises the selection of the proper marker/colour combination and dye concentration.

One should also take care about a proper control setup. The instrument itself requires particular care to ascertain that results are comparable between identical experiments. This is accomplished by standardization beads which can be used for daily instrument performance check. Moreover these data may serve to estimate the absolute number of molecules on the cell.

While using several fluorescent dyes simultaneously (polychromatic cytometry) one has to compensate the excessive signals resulting from the spill-over (overlap) of the dyes emitting light to adjacent channels. This is done to exclude fault positive events in other channels.

Another type of control which should be applied in multicolour staining for proper gating and to distinguish between positive and negative population is called fluorescent minus one (FMO) control which will be explained in detail. Finally, the last type of control consists of the samples fully stained but untreated which applies for experiments with cell stimulation.

If experiment is accurately designed and performed the errors are reduced and give a good starting point for further data analysis.

The above mentioned rules and standards are now being introduced by ISAC (International Society for Advancement of Cytometry) which is becoming a common practice for data acquisition, analysis and presentation.

Abstract no. L28

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HIGH-CONTENT ANALYSIS OF OXYGEN REACTIVE SPECIES AND OXIDATIVE STRESS

Pinto S, Gomes A, Herrera G, Díaz L, Parra M, Callaghan R, and O'Connor J
Laboratory of Cytomics, Mix Unit CIPF-UVEG, Centro de Investigación Príncipe Felipe and Universidad de Valencia

In vitro toxicity may predict drug adverse reactions and be useful to assess the toxic risk of chemical compounds. High-content assays (HCA) based on single-cell, with multi-parametric fluorescence measurements of heterogeneous systems, are new alternatives for analysis of cytotoxicity in vitro. Oxidative stress is a key process of in vitro toxicity as it may be provoked by many xenobiotics as well as it may arise from endogenous or induced alterations in oxygen metabolism. In vitro, cell-based analysis of oxidative stress biomarkers should ideally include the detection of the short-lived reactive oxygen species (ROS), the assessment of antioxidant defences and the demonstration of longer-term oxidative damage to intracellular components, such as DNA, proteins and membrane lipids. We present here the preliminary results of our own battery of HCA for oxidative stress assessment, suitable for studies of in vitro toxicity related to the sequential effects of prooxidant xenobiotics. Appropriate fluorescent dyes or fluorogenic substrates were used to assess superoxide, peroxides, glutathione, and the oxidized DNA base 8-oxoguanine, in cells treated with vehicle or positive control compounds. All assays have been performed on human cell lines in the automated epifluorescence microscope In Cell Analyzer 1000 and In Cell Developer Toolbox software for cell segmentation and data calculation. For validation, similar assays were run in parallel using flow cytometry. HCA correlates functional responses to morphological and structural parameters in oxidative toxicity studies. In addition our results reveal cell population heterogeneity and provide tools to address this phenomenon. Moreover, the 96-well format of HCA enhances data generation while lowering the number of cells per experiment. Sponsored by the EC Project A-Cute-Tox, LSHB-CT-2004-512051, and the Spanish Ministry of Science (BIO2007-65662).

Abstract no. L29

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IMPEDANCE MICROFLOW CYTOMETRY - PRINCIPLES, APPLICATIONS & OUTLOOK

Di Berardino M
Leister Process Technologies, Axetris Division

Impedance-based flow cytometry is not really a new inspiration for characterising particles or cells. The measurement of the electronic cell volume or Coulter volume in the 1950-ies was in fact one of the first cell parameters measured using a flow cytometric approach. Compared to the achievements of fluorescence-based flow cytometry, single cell impedance analysis provides with its enumerating and sizing capability only rather limited information content. The advent of micro-fabrication technologies in the last decade, however, promises a boost in sensitivity related to that of macro-scale impedance devices, enabling thereby true single cell characterisation. This tutorial will present a novel microflow cytometer, which is based on a microfluidic impedance chip. It will emphasize the potential of impedance microflow cytometry as a means of characterising cells without the need of fluorochromes or other dyes. In contrast to FACS instruments, which require specific cell markers for multiparametric cell analyses, this cytometer performs impedance measurements of single cells at various frequencies simultaneously. The resulting impedance signal provides information about cell volume, membrane capacitance and cytoplasmic conductivity, parameters that are directly related to the physiological conditions of single cells. Data obtained with different cell models indicate that the system can be a valuable alternative to conventional fluorescence-based flow cytometers for various applications in the field of cell differentiation or parasitology. Due to its high sensitivity but missing specificity, however, the presented technology is most suitable for routine and quality control analyses, such as viability studies for any kind of cells.

Abstract no. L30

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FLOW CYTOMETRIC DATA ANALYSIS WITH FLOWJO

Vallan C
Celeza GmbH

FlowJo is the fastest and most versatile program available for flow cytometry analysis. The philosophy of the developers to consider an experiment as more than a mere collection of single samples led to procedural features that allow an effortless analysis of very complex issues. Intricate analyses are quickly batched to dozens, hundreds or thousands of samples, still keeping it very simple to individually adjust the analyses on each one of the single specimens. Of course the characteristics of the program benefit also the analysis of “simple” experiments. The tutorial will give an introduction on how to operate FlowJo and on how to apply its unique features. Examples will show how to batch analyses on several samples, how to export the results from tables and layouts, how to create reports and how to set up templates that will facilitate your daily work. Moreover a short overview on the tools available will be given. This includes software compensation, proliferation analysis, cell cycle analysis, kinetics, quantitation, statistical comparisons and much more.

Abstract no. L31

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CELL SORTING

Lösche A
IZKF, University of Leipzig

Research involving cell analysis frequently requires isolation of certain cell types or subcellular components either as a final objective or as a preparative tool for further assays. At present, there are a high number of cell sorting methods that are suitable for use in the clinical laboratory. These methods can be divided into two major groups: non-optical and optical cell sorting methods.

Non-optical methods use magnetic fields (MACS) or differences in particle buoyancy and density. Magnetic cell sorting uses antibody-coated (magnetic) particles that bind to a specific cell type. However, magnetic cell sorters are unable to sort cells on the basis of cell size, morphology or intracellular markers.

Optical flow sorters measure and select user-defined cell types by illuminating individual cells with focused light and detecting the emitted light (Fluorescence-Activated Cell Sorting – FACS). Electrically-charged droplets are then produced containing the cells of interest and are deflected into collection tubes or into a well plate for further use.

As a consequence of the mechanisms underlying these two cell sorting methods, the balance between cell purity and cell recovery of the sorted fraction are generally different. The optical method (FACS) usually provides both a higher purity and recovery. Thus, in practice, non-optical methods are frequently used as a preparative step for optical cell sorting.

This tutorial will present the basics of the Fluorescence-Activated Cell Sorting (FACS - optical cell sorting method) in order to provide an understanding of the basic principles behind the technology, the capabilities and the limitations of the optical cell sorting.

POSTER

Abstract no. P01

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OBSERVATION OF THE APPEARANCE OF SEVERE B CELL LYMPHOMA IN FULLY IMMUNOCOMPETENT C57BL6 TRANSGENIC MICE, EXPRESSING PHYSIOLOGICALLY OVALBUMIN SPECIFIC T CELL RECEPTOR ON EITHER CD3 CD8 T SUPPRESSOR CELLS (OT-I) OR ON CD3 CD4 T HELPER CELLS (OT-II)

Arndt K,1 Wenk K,1 Edelmann G,2 Madaj-Sterba P,3 and Braun JM2
1Institut für Klinische Immunologie und Transfusionsmedizin, Universität Leipzig 2Translational Centre for Regenerative Medicine, Universität Leipzig 3Medizinisch Experimentelles Zentrum, Universität Leipzig

Transgenic mice physiological expressing ovalbumin specific T cell receptors are powerful models for the assessment of the function of regulatory and effector T cells in vivo. Transgenic animals express their T cell receptor (TCR) specific for a single antigen (here ovalbumin) on between 60 to 95% of all T cells in peripheral blood, thymus, and spleen, compared to wild type animals showing a rate of below 1% of TCR with a single antigen specificity.

Ovalbumin specific TCR (CD3 CD4) expressing animals are well established in mice with the BALB/c background (DO11.10). A new animal model of transgenic mice expressing physiologically ovalbumin-specific T cell receptor transgene mice on CD3 CD8 T suppressor cells (OT-I), or CD3 CD4 T helper cells (OT-II) were recently established and introduced to Leipzig for a small breeding programme.

Within the breeding of the first 20 animals for both strains, two cases and one case of severe B cell lymphoma were observed in both OT-1 and OT-II mice, respectively. Clinical symptoms included apathy, rough fur, a swelling of the abdomen due to an up-to 10 fold increase in spleen (5.5 x 0.75 cm) and liver size, massive swelling of all major lymph nodes up to a diameter of 0.4 cm, and a 5 fold increase in thymus size.

Peripheral T cell, monocyte, and granulocyte count was normal as assessed by flow cytometry, while both animals suffered from a up-to 20 fold increase in blood B cell count. Serological tests against viral and bacterial antigens tested were negative (MHV, Reo3, TMEV, PVM, Sendai, MVM, MPV, Rota, Mycopl. pulmonis, Past. pneumotropia).

OT1 and OT2 mice might provide a challanging animal model in oncology.

Abstract no. P02

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DETECTION OF PROANGIOGENIC CYTOKINES USING CBA TECHNIQUE IN A SMALL VOLUME OF SERUM AND PERITONEAL FLUID SAMPLE FROM WOMEN WITH ENDOMETRIOSIS, UNEXPLAINED INFERTILITY AND OVARY CYSTS

Baurska H,1 Jalocha I,2 Gabrys M,2 and Chrobak A1
1Faculty of Biotechnology, University of Wroclaw, 2First Department of Gynecology and Obstetrics, Medical University Wroclaw

Background: Endometriosis is a condition where endometrium, which normally lines the uterus, is found outside this organ. Pathogenesis of endometriosis and additionally a non-invasive method of its diagnosis are still unknown. There is some evidence that the immunological environment and angiogenesis play an essential role in the development of the disease. In our experiments with the use of flow cytometry we have measured in one small sample in a short time the level of five pro-angiogenic cytokines: angiogenin, VEGF, bFGF, TNF alpha and IL-8 in women with endometriosis in comparison to the control group.

Material and methods: In serums and peritoneal fluids harvested from 30 women with endometriosis, 10 women with unexplained infertility and 17 women with ovary cysts (control group) measure of pro-angiogenic proteins was done with the use of BD CBA Flex Set test. The cytokines levels were detected by FACS Calibur flow cytometer and data were analyzed using FCAP ArrayTM Software, BD.

Results: Concentrations of IL-8 in peritoneal fluids from women with severe stage of the endometriosis was higher (31,29 ± 5,00 pg/ml), than from women with mild endometriosis (9,89 ± 1,29 pg/ml). The differences were significant using the Kruskal-Wallis test when p-values were less than 0.05. We have also observed a similar tendency in serum. The level of VEGF in serum was lower (31,85 ± 1,5 pg/ml) in women with endometriosis in comparison to the control group (52,46 ± 1,43 pg/ml). We have also observed a similar tendency in peritoneal fluids.

Discussion and conclusions: Changed VEGF and IL-8 levels could influence an abnormal growth and development of endometrium in women with endometriosis.

Abstract no. P03

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PLATELET-ACTIVATING FACTOR- STIMULATED PRODUCTION OF REACTIVE OXYGEN SPECIES IN OVARIAN GRANULOSA CELLS FROM PREOVULATORY FOLLICLES

Xu J,1 Krüger B,2 Vernuft A,1 Löhrke B,3 and Viergutz T1
1FBN-Dummerstorf 2Faculty of Medicine, University of Rostock 3FBN-Dummmerstorf

Background: The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood.

Methods: Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine preovulatory follicle. Expression of PAFr mRNA and PAFr protein was assessed by RT-PCR and immunochemistry. Cytosolic calcium (Ca) concentration was assayed by microscopy using Fura-2 AM as indicator, progesterone and 8-isoprostaglandin F2alpha (iPG) by RIA and an ELISA kit. Fluorescent products arising from intracellular oxidation of dihydroethidine and dihydrorhodamine were quantified by flow cytometry.

Results: The cells expressed PAFr mRNA and PAFr protein and responded to PAF with a pulsating increase in Ca, demonstrating functional PAFr. Elevation of Ca was reversed by WEB-2086, an inverse PAFr agonist, indicating specific data. PAF elevated the level of iPG in the medium of cultured MGC from healthy follicles larger two times the control. Despite weak PAF-induced oxidation of non-fluorescent precursors, fluorescent products corresponded to iPG level.

Conclusion: In MGC from vital preovulatory follicle, PAF signaling plays an important regulatory role in the production of reactive oxygen species. Blockage of PAFr seems to impair ovarian activity through interference with this PAF-mediated regulation.

Abstract no. P04

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CYTOTOXIC ACTIVITY AND INFLUENCE ON PHYSIOLOGY OF IMMUNE SYSTEM OF SELECTED ANTIMICROBIAL PEPTIDES

Pierzchalski A,1 Golab K,2 Kamysz W,3 and Tarnok A4
1Translational Center for Regenerative Medicine, University of Leipzig 2Intercollegiate Faculty of Biotechnology, University of Gdansk3Department of Inorganic Chemistry, Medical Universityof Gdansk 4Department of Pediatric Cardiology, Heart Center Leipzig, University of Leipzig

Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system for defense against invading pathogens. In addition, it was proved that AMPs possess mitogenic, anti-tumour and anti-viral activity. These activities imply usefulness of AMPs as potential therapeutic agents. The aim was to test synthetic AMPs for their immunomodulatory properties. Eleven antimicrobial peptides were synthesized.Their biological activity on isolated subpopulations of human white blood cells (lymphocytes, neutrophiles) was investigated by flow cytometry.

The AMPs analyzed displayed dose- and time-dependent cytotoxic effects. Analogs of AMPs of natural origin appeared to be less cytotoxic than synthetic ones, as was revealed in the viability tests performed with propidium iodide staining. Screening of immunomodulatory properties of examined peptides was done by testing cell physiological parameters: pH, Ca2, transmembrane potential, oxidative metabolism after preincubation with the compounds and then stimulation of the isolated leukocyte subpopulations with PMA, fMLP or tuftsin. Our studies showed that the examined AMPs directly did not affect physiological functionality of immune cells.

Due to the fact that some endogenous AMPs have been found to stimulate cell-mediated immunity by recruiting leukocytes, the abilities of the egzogenous peptides to stimulate the immune cells should be furthered performed thus determining the immunomodulatory effect.

Abstract no. P05

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ENZYMATICALLY DEGRADED LDL (E-LDL) AND OXIDIZED LDL (OX-LDL) INDUCE DIFFERENT LIPID DROPLETS IN HUMAN MACROPHAGES AS DETERMINED BY HIGH CONTENT SCREENING USING BODIPY 493/503

Grandl M, and Schmitz G
University of Regensburg

Background: The cholesterol-loaded macrophage foam cells after atherogenic lipoprotein uptake are a hallmark of atherosclerotic lesions and contribute to lesion development. E-LDL is taken up by macrophages through type I and type II phagocytosis while Ox-LDL is taken up through scavenger receptors. The different uptake mechanisms influence lipid storage, degradation and cellular functions.

Material and Methods: To analyse lipid droplets in human macrophages incubated with the atherogenic lipoproteins E-LDL and Ox-LDL a fluorescence-based in vitro high content screening assay was developed. After MCSF differentiation and 24h of lipoprotein incubation cells were fixed with paraformaldehyde and lipid droplets were stained using Bodipy 493/503. Images of 96-well cell-culture microtiter plates were captured with multi channel laser based confocal microscopy and subsequently analyzed. Intensity of Bodipy 493/503 staining was quantified and lipid droplet size and volume was determined.

Results: E-LDL leads to increased intensity of Bodipy 493/503 staining and a higher number of lipid droplets per cell. Also characteristically larger lipid droplet areas and volumes have been observed in E-LDL loaded macrophages compared to MCSF differentiated and Ox-LDL loaded macrophages.

Discussion and Conclusion: The different lipid droplet induction upon E-LDL and Ox-LDL loading indicates a differentially influence on lipid storage in macrophages leading to diverse effects on the pathogenesis of atherosclerosis. This assay represents the differential uptake mechanisms of E-LDL and Ox-LDL. While uptake of E-LDL leads to increased lipid droplet formation, Ox-LDL uptake leads to rapid expansion of the lysosomal compartment which is probably accompanied by increased unesterified cholesterol content in the lysosome. This assay provides a useful tool to study lipid droplets in foam cells under drug treatments affecting lipid droplet formation and storage.

Abstract no. P06

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DNA FLUORESCENCE AS TRIGGER FOR SCANNING FLUORESCENCE MICROSCOPY (SFM)

Mittag A,1 Marecka M,2 Malkusch W,3 Pierzchalski A,2 Bocsi J,2 and Tárnok A2
1Translational Centre for Regenerative Medicine (TRM Leipzig), University Leipzig, Leipzig, Germany 2Dept. of Pediatric Cardiology, Heart Center, University Leipzig, Leipzig, Germany 3Carl Zeiss Imaging Solutions GmbH, Zeiss Group, München, Germany

Background: DNA staining is a common trigger signal for cell identification in imaging as well as in flow cytometry. However, selection of proper DNA dyes is mainly restricted by hardware configurations (i.e. detection filters or excitation sources) of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope – SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of this study was to select optimal DNA dyes for triggering in leukocyte samples and subsequent cytometric analysis of double-labeled leukocytes by SFM.

Methods: Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 20, fs: 44, fs: 49, fs: 50). EDTA blood was stained after erythrocyte lysis with one of the mentioned DNA dyes. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst.

Results: Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements.

Conclusion: DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

Abstract no. P07

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IN VITRO WOUND HEALING MODELS TO STUDY FIBRIN SEALANT

Wiesner C, Bunjow C, Einsenberger K, Pflüger M, Eger A, Hundsberger H, and Schütt W
IMC Fachhochschule Krems, Piaristengasse 1, 3500 Krems, Austria

To test fibrin sealants for their effect on wound healing, new methods for in vitro testing were developed in multi-well-chip-structures. In addition to human mono- and co-cultures, newly established 3D skin-systems are being used to determine the regenerative potential of fibrin sealants. Therefore the technology of Electric Cell-Substrate Impedance Sensing (ECIS) was adapted to detect and analyse the composition of a 3D skin model with high temporal resolution and without the addition of markers. Alongside the ECIS technology new automated and standardized methods utilizing a multi-plate reader are established and optimized for high throughput screening, resulting in faster and cheaper screenings of active agents and good reproducibility of the achieved results. This newly established model allows detection of the phases of wound healing, providing an ideal tool for high throughput screening of substances promoting wound healing.

Abstract no. P08

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TRANSPLANTATION OF ALLOGENEIC HEPATOCYTE IN THE RAT

Brückner S,1 Dollinger M,2 Stock P,3 Hempel M,4 Ebensing S,4 Mittag A,5 Tarnok A,5 and Christ B4
1KIM I, AG Molekulare Hepatologie, Uniklinikum Halle, 2KIM I Uniklinikum Halle 3KIM I, AG Molekulare Hepatologie, Uniklinikum Halle, 4KIM I, AG Molekulare Hepatologie, Uniklinikum Halle, 5Herzzentrum Leipzig

Background: The immune response leading to rejection of transplanted hepatocytes has not yet been addressed in detail. In the study presented, the rate of hepatic repopulation by transplanted hepatocytes in a syngeneic model (donor: DPPIV/F344; recipient: DPPIV-/- F344) was compared to an allogeneic transplantation model (donor rat: Dark Agouti; recipient rat: DPPIV-/- F344).

Material and method: Wildtype donor hepatocytes delivered via portal injection were detected by histochemical staining of DPPIV enzyme activity in the otherwise negative host liver background and quantified by flow cytometry.

Results: 24 hours after transplantation, single hepatocytes were detected in the recipient liver parenchyma both in the syngeneic and allogeneic model. 5 days post-transplantation, small clusters of 1-5 donor hepatocytes were visible in the syngeneic model as compared to still single cells in the allogeneic model. After 3 weeks, in the syngeneic model 1 % of the recipient liver was replaced by donor hepatocytes while no cells were found in the allogeneic model. Depletion of Kupffer cells (KCs), macrophages resident in the liver, by gadolinium chloride did not increase the rate of repopulation in either model.

Discussion and conclutions: Thus, KCs mediating the early immune response, were not involved in the rejection of allogeneic hepatocytes, but rather T-cells eliminating transplanted allogeneic hepatocytes during a three weeks period in time.

Abstract no. P09

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PHASE CONTRAST SIGNAL FOR TRIGGERING OF LEUKOCYTES IN IMAGING CYTOMETRY WITH SCANNING FLUORESCENCE MICROSCOPE (SFM)

Bocsi J,1 Pierzchalski A,1 Marecka M,1 Malkusch W,2 and Tárnok A1
1Heart Center, University of Leipzig, Pediatric Cardiology, Leipzig, Germany 2Carl Zeiss, Imaging Solutions GmbH, Munich Hallbergmoos, Germany

Innovative slide-based cytometry (SBC) systems lead to breakthrough in cytometry as they provide sophisticated tools for analyses of cells in tissues, culture and suspension. Zeiss Imaging Solution GmbH introduced a new automated fluorescence microscope that combines imaging with cytometric features (acquiring high number of quantitative data from individual cells in multiple fluorescence channels). A critical step in image analysis is selection of appropriate triggering signal to detect all objects. Without correct target cell definition analysis is hampered. DNA-staining is among the most common triggering signals. However, the majority of DNA-dyes yield massive spillover into other fluorescence channels limiting their application. By microscopy, objects of >5μm diameter can be easily detected by phase-contrast signal (PCS) without any staining. Aim was to establish PCS - triggering for cell identification.

Methods: Axio ImagerZ1 fully motorized microscope (SFM) AxioCam MRm and AxioVision software (suitable for automatic multichannel scanning and fluorescent analysis) were used. Samples were scanned in three channels (PCS, FITC, Cy5). Beads and mononuclear cells were used. Cells were stained by antibodies labeled with FITC APC or Alexa647 in different combinations. Leukocytes stained with anti-CD45 antibodies were counted and the result was compared with the amount of PCS detected events. The proportion of leukocyte subpopulations measured with PCS as trigger signal was compared with LSC and FCM data.

Results: Focused phase contrast signals showing ring form were not optimal for the cell definition. PCS slightly out of focus allows for effective qualitative and quantitative cell analyses. Cell count triggered by PCS and CD45 was highly correlated (P=0.0003, R=0.809). Leukocyte subpopulation measurements triggered on PCS were comparable with leukocyte subpopulation distribution performed with LSC and FCM.

Conclusion: PCS is a suitable trigger-signal for bead and leukocyte detection thus enabling cell counting and discrimination of leukocytes from platelets and it is not interfering with fluorescence detection.

Abstract no. P10

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MONOCYTIC DIFFERENTIATION OF VARIOUS ACUTE MYELOID LEUKEMIA CELL LINES INDUCED BY SIDE-CHAIN MODIFIED SEMI-SELECTIVE ANALOGS OF 1,25-DIHYDROXYVITAMIN D3

Baurska H,1 Kutner A,2 and Marcinkowska E1
1Faculty of Biotechnology, University of Wroclaw, 2Pharmaceutical Research Institute, Warszawa

Background: 1,25-Dihydroxyvitamin D3 (1,25D) mediates its biological effects by binding to the vitamin D receptor (VDR) which acts as a transcription factor. In addition to regulation of calcium homeostasis 1,25D is involved in immunomodulation, cell proliferation and differentiation. Thus differentiation therapy of myeloid leukemia using 1,25D is a very attractive alternative to existing treatments, but undesirable side effects of 1,25D caused that its semi-selective analogs, with reduced calcemic activity, have been synthesized. Usually HL60 acute myeloid leukemia (AML) cell line has been used for selection of candidate analogs for future studies. Here we used other leukemic cell lines: NB-4, THP-1 and U937 in order to compare activities of semi-selective side-chain modified 1,25D analogs named: PRI-1906, PRI-2191, PRI-2201 and PRI-2202.

Material and methods: The expression of cell surface markers CD11b and CD14 was analyzed in flow cytometry.

Results: We have noticed that expression of monocytic differentiation marker CD14 is upregulated in response to 1,25D or its analogs stronger than expression of CD11b, which is a marker of monocytic and granulocytic differentiation. Out of the three cell lines studied, differentiation was the strongest in THP-1 cell line. The differentiation-inducing potency of PRI-1906 and PRI-2191 was comparable to that of 1,25D in THP-1 cells and higher in NB-4 and U937 cells.

Discussion and conclusions: Our experiments point at a variable susceptibility to 1,25D-induced cell differentiation of cells derived from various subtypes of leukemia, and show that the analogs PRI-1906 and PRI-2191 are the most promising candidates for antileukemic therapy.

Keywords: 1,25-dihydroxyvitamin D3, side-chain modified analogs, differentiation, THP-1, NB-4, U937, flow cytometry

Abstract no. P11

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UV LED EXCITED TIME-RESOLVED FLUORESCENCE MICROSCOPE

Connally R, Dayong J, Piper J, and Lawson T
Centre for Lasers and Applications, Macquarie University, Sydney

Many naturally occurring substances are intrinsically fluorescent (autofluorescent) when exited at an appropriate wavelength and emission can occur throughout the visible spectrum. Autofluorescence is typically a short-lived phenomena with a lifetime (t) measured in nanoseconds and this property is exploited in Time-Resolved Fluorescence (TRF) microscopy to enhance detection of labelled pathogens against autofluorescence background. The TRF methods are based on the use of immunofluorescent labels with long fluorescence lifetimes (∼600 μs) to ensure that labelled target is viable long after short-lived autofluorescence has faded. Pulsed excitation is used to excite fluorescence from the sample and this is followed by a gate-delay phase to permit decay of short-lived fluorescence. When flashlamps are used as the excitation source, the gate-delay period must be extended (>50 μs) to ensure that light from the decaying plasma has decayed to zero. The extended gate-delay results in a significant loss of fluorescence intensity from the synthetic label and this is avoided with solid-state excitation sources. A high-power (>100 mW) Light Emitting Diodes (LEDS) (λ 365 nm) was substituted for the flashlamp and found to give excellent background suppression and strong label fluorescence compared to flashlamp excitation.