DUBLIN GUINNESS STOREHOUSE - IRISH CYTOMETRY SOCIETY
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6 ANNUAL MEETING
TH
OF THE
IRISH CYTOMETRY SOCIETY
17TH – 18TH NOVEMBER 2010
GUINNESS STOREHOUSE
DUBLIN
WEDNESDAY, NOVEMBER 17 TH
8:30 – 9:30 Registration
9:30 – 9:40 Welcome Barry Moran Irish Cytometry Society
Session 1: Immunology and Cytometry
Joint Chair: Peter O’Toole and Barry Moran
9:40 – 10:20 Rhodri Ceredig National University of Ireland, Galway
Use of flow cytometry to study lymphocyte development: Fac(t)s and aretfac(t)s
10:20 – 10:40 James Harris Trinity College Dublin
Autophagy suppresses IL‐1β secretion by targeting pro‐IL‐1β for degradation in macrophages and
dendritic cells
10:40– 11:20 Presentation generously sponsored by Medical Supply Company
Derek Doherty Trinity College Dublin
All sorts of iNKT cells
11:20 – 11:50 Poster Session and Break
11:50– 12:10 James Corcoran University College Dublin
IHG‐1, a Mitochondrial Protein, Regulates Mitochondrial Biogenesis and Mitochondrial Network Integrity
12:10 – 12:40 Presentation generously sponsored by Millipore
Christian Muller Université de Strasbourg
Capillary cytometry for a one‐stop‐shop HCS: Cytomic studies by cell based assays followed by integrated
multi‐dimensional analysis for the assessment of inflammatory disease therapies
12:40 – 13:00 Alfonso Blanco University College Dublin
Measurement of physiological changes. Improvements on a continuous flow cytometric methodology
13:00 – 14.00 LunchSession 2: Advancements in Technology Joint Chair: Rhodri Ceredig and Alfonso Blanco 14:00 – 14:40 Yuri Volkov Trinity College and Institute of Molecular Medicine, Dublin Nano‐reporters and nano‐bullets: how nanotechnology advances enable to probe and target human cellular structures? 14:40 – 15:00 Jennifer Dorney Dublin Institute of Technology Polystyrene: A potential standard for developing In Vitro cellular tracking methods for nanotoxicology. 15:00 – 15:40 Peter O’Toole University of York, UK Super Resolution Microscopy ‐ a new approach 15:40 – 16:00 Poster Session and Break 16:10 – 16:50 Hugh Byrne Dublin Institute of Technology Understanding the Interaction of Nanoparticles with Biological Cells 16:50 – 17:10 Jong Ah Kim University College Dublin Effect of cell cycle on nanoparticle uptake 17:10 – 17:30 Sergio Anguissola University College Dublin Establishment of a High Content Analysis (HCA) platform to assess nano‐toxicology and explore nanoparticle‐induced cell death 17:30 – 18:30 Happy Hour!
THURSDAY, NOVEMBER 18 TH Session 3: Clinical Cytometry Joint Chair: Niga Nawroly and Sean Rooney 9:30 – 10:00 David O’Brien St. James’ Hospital, Dublin. Immunophenotyping, its role in Acute Leukaemia 10:00 – 10:20 Egle Passante The Royal College of Surgeons in Ireland, Dublin A high throughput flow cytometry assay to screen the cytotoxicity of dual‐drug combinations 10:20 – 11:00 Andy Rawstron Leeds Teaching Hospitals, UK To be announced (Current Issues in Lymphoma Diagnosis) 11:00 – 11:30 Poster Session/Coffee Break 11:30 – 11:50 Susan Kennedy University College Dublin Determining the role for surface markers of neutrophil migration as predictors of post‐operative clinical outcome following open heart surgery 11:50 – 12:30 David Bloxham Addenbrooke’s Hospital, Cambridge University Hospitals, UK Polychromatic flow cytometry in clinical applications… pause for thought 12:30 – 12:40 Alfonso Blanco Irish Cytometry Society ESCCA 2011 in Dublin 12:40 – 13:10 Caroline MacKell ICON Plc., Dublin Accreditation in the flow cytometry lab – the ICL perspective 13:10 – 14.00 Lunch
Session 4 Emerging Applications of Cytometry
Joint Chair: Barry Moran and Sergio Anguissola
14:00 – 14:40 Markus Rehm Royal College of Surgeons in Ireland, Dublin
Biophotonic and Systems Biological Investigation of Cell Death Signaling
14:40 – 15:00 John Tigges, Vasilis Toxavidis Harvard University, USA
New Advances in Flow Cytometry Software
15:00 – 15:30 Niga Nawroly National Heart and Lung Institute, Imperial College London, UK
Multicolour flow cytometry analysis in RSV infection
15:30 – 16:00 Poster Session and Break
16:00 – 16:20 Simrat Kaur National University of Ireland, Galway
Flow cytometric studies in microalgae
16:20 – 16:50 Presentation generously sponsored by Partec
Jane Walker Scotch Whiskey Research Institute, Edinburgh, UK
Monitoring Fermentation Performance of Distilling Yeast (Saccharomyces cerevisiae) During Scotch
Whisky Fermentation using Flow Cytometry
16:50‐17:00 Conclusion; and Oral, Poster and Raffle Presentation Prizes
Alfonso Blanco Irish Cytometry SocietyGOLD SPONSORS
SILVER SPONSORS
BRONZE SPONSORS FLYER SPONSORS
ABSTRACTS Use of flow cytometry to study lymphocyte development: Fac(t)s and aretfac(t)s Rhodri Ceredig Regenerative Medicine Institute, National University of Ireland, Galway My talk will focus on the contribution of flow cytometry to our understanding of thymocyte development. The advent of flow cytometry and monoclonal antibody technology in the late 1970’s resulted in a revolution in our understanding of haematology and immunology. Initially, based on a variety of parameters, including cell surface marker expression by antisera and lectins, sensitivity to glucocorticoid ablation and cell size, numerous models of mouse thymus development had been proposed. However, using xenogeneic monoclonal antibodies (mAb) to human lymphocyte subsets, the seminal papers from Reinherz and Schlossman in the 1980’s correctly identified the CD4/CD8 model of thymocyte development we accept today. In mouse, the CD4/CD8 model had to wait the serendipitous generation in 1982 of the first anti‐mouse CD4 mAb GK1.5. Using combinations of surface marker and cell cycle analysis, the demonstration that cells expressing neither CD4 nor CD8 antigens, so‐called double negative (DN) cells, could differentiate in vitro to cells expressing both markers began studies that further dissected the inter‐relationships between thymocyte subpopulations. The ability to isolate DN cells led to the demonstration that some expressed the alpha chain of the Interleukin‐2 receptor complex, the CD25 antigen. Discovery of the T cell receptor genes and the resultant molecular analysis of the rearrangement status within thymocyte subsets confirmed the major pathways of T cell development in the thymus. From this emerged in 1993 the Godfrey and Zlotnik model of early (DN) mouse thymocyte development. Much effort was then made into identifying the rare (approximately ten cells per day in the mouse) bone marrow‐derived progenitor cells that must continually seed the thymus in order to maintain thymopoiesis. This pathway of discovery is still ongoing but has been greatly assisted by in vitro culture systems whereby hematopoiesis and thymopoiesis can be studied using in vitro stromal cell‐based culture systems. Identification of additional “non‐conventional” T cell subpopulations within the thymus, such as “natural‐killer T” (NKT), “natural regulatory T” (TReg) and T cells expressing gamma/delta T cell receptor heterodimers (γδT) has added further complexity to our understanding of thymocyte development. Experiments combining the phenotypic analysis of rare subpopulations of cells with transcriptional profiling bring with them increasing demands on sophisticated flow cytometric labelling and sorting. Some issues related to this will be discussed. Autophagy suppresses IL‐1β secretion by targeting pro‐IL‐1β for degradation in macrophages and dendritic cells James Harris & Ed Lavelle Trinity College Dublin
Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen‐associated molecules and has recently been shown to influence IL‐1 secretion by macrophages. However, the mechanisms behind this are unclear. The aim of this study was to further delineate the nature of the interactions between autophagy and the inflammasome system and here we describe a novel role for autophagy in regulating the production of inflammatory cytokines in antigen‐presenting cells. After treatment of macrophages with Toll‐like receptor (TLR) ligands, pro‐IL‐1β was specifically sequestered into autophagosomes and further activation of autophagy with rapamycin or starvation induced the degradation of pro‐IL‐1β and blocked secretion of the mature cytokine. Inhibition of autophagy with 3‐ methyladenine (3‐MA), wortmannin or siRNA against beclin 1 (Atg6) promoted the processing and secretion of IL‐1β by LPS‐stimulated antigen‐presenting cells in a NLRP3‐ and TRIF‐dependent manner. This effect was reduced by inhibition of reactive oxygen species, but was independent of NOX2. Induction of autophagy in mice in vivo reduced serum levels of IL‐1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL‐1β by targeting pro‐IL‐1β for lysosomal degradation and regulating activation of the NLRP3 inflammasome. This represents a potentially pivotal role for autophagy in controlling the response to inflammatory stimuli. This work was supported by Science Foundation Ireland as part of the Immunology Research Centre, SFI Strategic Research Cluster. All sorts of iNKT cells Derek G. Doherty, Institute of Molecular Medicine, Trinity College Dublin, St. James’s Hospital, Dublin, Ireland Trinity College Dublin Invariant natural killer T (iNKT) cells are innate T lymphocytes that recognise glycolipids presented by CD1d. They have potent antitumour activities. Therapeutic activation of iNKT cells with the agonist glycolipid α‐galactosylceramide (α‐GalCer) can prevent and reverse tumour growth in murine models. However, clinical trials involving these cells in humans have had limited impact. This might be because murine and human iNKT cells have different glycolipid specificities or functions or because there are different subsets of iNKT cells and the “wrong” subsets were used in the clinical trials. We have addressed these questions by using a variety of flow cytometry and cell sorting techniques to phenotypically and functionally characterize human iNKT cells isolated from various organs. We show that iNKT cells are found at ~100‐fold lower numbers in blood, liver and gut than in the corresponding locations in mice and that they display different cytokine secretion profiles. However, iNKT cells are uniquely abundant in human omentum. Using expanded lines and clones of human iNKT cells, we have demonstrated that these cells are capable of multiple cytokine production, cytotoxicity, inducing maturation of dendritic cells into antigen‐presenting cells and inducing maturation of B cells into antibody‐secreting plasma cells. We also compared the functions of CD4+, double‐negative (DN) and CD8+ iNKT cells and found that CD4+ iNKT cells secreted higher amounts of the Th2 cytokines IL‐4 and IL‐ 13 whereas DN and CD8+ cells secreted more IFNγ. CD8+ iNKT cells displayed most potent cytotoxic activity against CD1d+ target cells. These results highlight a potential role for CD8+ iNKT cells over CD4+ and DN iNKT cells in antitumour immunity. They indicate that iNKT cells are heterogenous in nature and have distinct distributions and functions in different organs of the body. Future therapeutic trials for cancer treatment may benefit from using selected subtypes of iNKT cells.
IHG‐1, a Mitochondrial Protein, Regulates Mitochondrial Biogenesis and Mitochondrial Network Integrity Fionnuala B Hickey, James Corcoran, Brenda Griffin, Una Bhreathnach, David Cottell, Finian Martin, Catherine Godson and Madeline Murphy Induced in high glucose‐1 (IHG‐1) is a novel gene that we have described to be upregulated in human diabetic kidney disease. To date a mitochondrial localisation sequence is the only predicted functional domain identified in IHG‐1. We have confirmed the mitochondrial location of IHG‐1 by fluorescent confocal microscopy following over expression of V5‐tagged IHG‐1. Immunogold cryosection electron microscopy has indicated that IHG‐1 is located predominantly in the mitochondrial matrix and is associated with the inner mitochondrial membrane. Mitochondrial dysfunction has recently been reported to be a major contributor to hyperglycemic‐ induced kidney damage and also has a well documented role in fibrosis. We have further investigated the role of IHG‐1 in mitochondrial function using a combination of flow cytometry, live‐cell imaging techniques and other methods. These analyses have been carried out in both cell lines that over‐express IHG‐1 and using shRNAi‐mediated knockdown of endogenous IHG‐1. Loss of IHG‐1 has been found to lead to decreased mitochondrial biogenesis. In addition, we have studied the effect of decreased IHG‐1 expression on mitochondrial dynamics using fluorescence recovery after photobleaching (FRAP) and other live cell imaging fluorescent techniques. Decreased mitochondrial fusion is seen in cells in which IHG‐1 expression has been knocked down. Interestingly, we have found that IHG‐1 expression is upregulated by reactive oxygen species (ROS). In addition, the effect of overexpression of IHG‐1 on oxidative stress has been measured by flow cytometry using the fluorescent probe H2DCFDA. A potential role for IHG‐1 in apoptosis has also been investigated using Annexin V / Propidium iodide staining and flow cytometry. Therefore, a combination of flow cytometry and fluorescent and electron microscopy has enabled us to characterise the location and function of a novel protein, IHG‐1, which may have a critical function in both diabetic nephropathy and other fibrotic conditions. Capillary cytometry for a one‐stop‐shop HCS: Cytomic studies by cell based assays followed by integrated multi‐dimensional analysis for the assessment of inflammatory disease therapies Christian Muller Université de Strasbourg Flow cytometry (FCM) is a fundamental and powerful tool for cellular analysis with applications for biotechnology spanning from basic research to clinical diagnostics. In flow, samples are evaluated on a per cell basis for size, shape, protein abundance (both surface and intracellular), and/or functional status. Thousands of different flow‐based assays have been validated for use on cells or with particles, ranging from single‐parameter endpoint tests to complex assays involving multiple cell populations or simultaneous analysis of multiple responses. While traditionally a manually‐loaded tool for sample‐by‐ sample analysis, recent advances have made possible the use of FCM in higher throughput experiments. However, data analysis has remained a time‐consuming activity requiring significant manual intervention for gating as well as for overall data reduction and interpretation. In this report, we demonstrate the use of a small semi‐automated capillary cytometer, with integrated plate‐based
analysis software, in screening a large library of small molecules for both pro‐apoptotic and anti‐ inflammatory properties. Incyte software offers simplified raw data analysis, experiment‐level results display for hit compound identification, and multiparameter comparative studies thereby allowing the researcher to rapidly query multiple statistical results for multiple wells (a 96‐well plate). Measurement of physiological changes. Improvements on a continuous flow cytometric methodology Alfonso Blanco University College Dublin The identification and measurement of morphological and physiological changes in cells through time is crucial for the understanding of the cell itself, as well as its interaction with media and other cells. For instance, calcium regulates a wide range of vital cell functions including signal‐transduction, electrochemical responses, enzyme activities, metabolic processes, cell‐cycle progression, replication, morphology, mobility etc... Some of these changes are amongst the most rapid responses in mammalian cells, and in some systems, like the nervous system or platelets, responses occur within nanoseconds. To date, the kinetics of these changes are monitored by microscopy, plate based assays, spectrofluorometry and flow cytometry, although there are issues with the number of cells analysed, or missing information due to the addition of compounds, with significant loss of detail of a rapid response. The Accuri C6 resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic morphological and physiological measurements, whilst also providing extensive and valuable data regarding population health and responsiveness. This method could be applied to any other flow cytometer with a peristaltic pump that allows continuous cell aspiration from an open sample tube or vial. With these new cytometers the cell response to a stimulus becomes extremely accessible, whilst also providing extensive and valuable data regarding population health and responsiveness. Comparative measurements using the Accuri C6, the stop‐flow methodology and systems such as the Cytex time‐zero module would provide additional insights into the scope of the Accuri C6. This methodology allows the easy measurement of the changes induced to cells in their morphology, signal‐transduction, electrochemical responses, enzyme activities, cell‐cycle progression, replication, phagocytosis, nanoparticle uptake and metabolic processes such pH, reactive oxygen, nitrogen species, mitochondrial membrane potential etc. Nano‐reporters and nano‐bullets: how nanotechnology advances enable to probe and target human cellular structures? Yuri Volkov, PhD, MD Trinity College and Institute of Molecular Medicine, Dublin, Ireland Nanomedicine as a rapidly expanding area of science provides a unique chance to exploit the diverse properties of engineered nanomaterials for the ultimate benefit of the patients. Light‐emitting nanoparticles, including fluorescently doped silica, polystyrene nanoparticles and quantum dots have an
outstanding potential for diagnostic applications and intracellular imaging in biomedical research. On the other hand, optimistic expectations are associated with the opportunities of using the nanoparticles as a new class of drug delivery systems, arising from the fact that the finite, but tunable size of the engineered nanostructures used as drug delivery vehicles can impose very precise nano‐scale drug distribution barriers at the level of cells, tissues and entire organism, thereby eliminating undesirable side effects pertinent to most contemporary medicines. However, there is still very little definitive systematic information about the consequences of interactions of nano‐scale objects with human cells of diverse origin and therefore safety‐related issues are high on the nanomedical agenda. Phagocytes, epithelium of the lungs and gastrointestinal tract as well as cells of the cardiovascular system are the primary candidates to encounter these nanomaterials. We will provide here an overview of several nanoparticle application scenarios for intracellular delivery and imaging in different cell types, along with the contemporary approaches to safety screening of nanomaterials with promising biomedical application potential. Polystyrene: A potential standard for developing In Vitro cellular tracking methods for nanotoxicology. Jennifer Dorney, Dr Alan Casey, Dr Gordon Chambers, Prof Hugh Byrne. Nanolab, Focas Institute, Dublin Institute of Technology. Nanotoxicology has emerged as a discipline of a result of the revolution of nanotechnology. While nanotoxicology is in its infancy, there is a lack of toxicological data for nanoparticles, naturally occurring or commercially produced. The need for information regarding cellular uptake mechanisms associated with nanoparticle uptake, as well as internalisation and accumulation of nanoparticles once penetrating cell membranes, is imperative. This project will focus on the internalisation studies of surface modified polystyrene nanoparticles. An in vitro lung model consisting of A549 (ATCC No: CRL185) a carcinogenic lung epithelial cell line, was employed to investigate the biocompatibility of nano scaled polystyrene particles in pulmonary systems. Bulk polystyrene particles (above 3 µm) were employed as positive control and biological effects were compared to that of 40nm carboxilated surface modified nanopolystyrene and 50nm neutral nanopolystyrene. Prior to cellular studies, a full particle size characterisation was carried out using Dynamic Light Scattering, Atomic Force Microscopy, Zeta Potential Electronic Spectroscopy. The cytotoxic effects of nano scale 40nm carboxilated and 50nm neutral nanopolystyrene were then evaluated using five cytotoxic endpoints namely the Neutral Red, Alamar Blue, Comassie Blue, MTT and Clonogenic assays, with bulk polystyrene employed as a control, with both particles exhibiting no cytotoxic effect at any of the concentrations or endpoints examined. Cellular internalisation of the fluorescently labelled particles was monitored with the aid of fluorescent confocal microscopy. Raman spectroscopy was employed as a novel technique for the verification of nanoparticles internalised within cells. Nanoparticle internalisation and accumulation was then monitored as a function of nanoparticle surface charge and verified with the aid of commercially available transfection labelling kits.
Super Resolution Microscopy ‐ a new approach Peter O’Toole University of York, UK Understanding the interaction of nanoparticles with biological cells Hugh J. Byrne Focas Research Institute, Dublin Institute of Technology In terms of concerns over potential human and environmental hazards, and also in terms of applications in the emerging field of nanomedicine, an understanding of the interaction of nanoparticles with biological cells is imperative. The presentation will describe efforts within DIT, in the framework of the Integrated NanoScience Platform for Ireland (INSPIRE) as well as the FP7 Concerted Action NanoImpactNet, to establish structure property relationships governing the interaction of polymeric nanoparticles with a range of human cell lines. Dependence on particle size, surface chemistry and cell line will be discussed. Confocal microscopy and Flow cytommetry are employed to help elucidate the mechanisms underlying commonly employed cytotoxicity assays. Effect of cell cycle on nanoparticle uptake Jong Ah Kim, Christoffer Åberg, Iseult Lynch, Anna Salvati, Kenneth A. Dawson Centre for BioNano Interactions, School of Chemistry and Chemical Biology and Conway Institute, UCD Nanoparticles hold great promise as drug delivery vehicles for cancer treatment, where the lack of drug specificity for malignant cells limits the efficacy of current treatments and often leads to undesired side effects. Different parameters are being studied in relation to nanoparticle uptake in order to assess their potential role in preferential targeting of cancer cells. Some of these parameters include physicochemical characteristics of nanoparticles such as size, material and surface modifications, but here we have also investigated how nanoparticle uptake is affected by biological parameters such as the phase of the cell cycle at which cells are when they are exposed to nanoparticles, and the cell density within the culture (which can affect cell‐to‐cell communication). We have found that particle uptake occurs regardless of the phase in which the cells are. Using a double‐staining technique that allows us to follow the cell cycle in real time by flow cytometry, we have measured the rate of nanoparticle uptake in the different cell cycle phases. In addition to this, we have modelled the uptake kinetics of nanoparticles taking into consideration the effects of cell division which distributes the particle load among the daughter cells. Moreover, we have observed that particle internalisation is also influenced by the stage of growth in which the cultures are at the moment of exposure to particles. Together these data provide valuable insights into a range of factors that can contribute to non‐homogenous distribution of nanoparticles across cells following exposure, and are enabling us to develop more accurate descriptors of actual cellular doses of nanoparticles in a cellular population.
Establishment of a High Content Analysis (HCA) platform to assess nano‐toxicology and explore nanoparticle‐induced cell death Sergio Anguissola1, Fengjuan Wang1, Sonia Ramirez1, Anna Salvati1, Peter O’Brien2, Kenneth Dawson1 1 Centre for BioNano interactions, School of Chemical Biology, University College Dublin (UCD), Belfield, Dublin 4, Ireland. 2 College of Life Sciences, School of Agriculture, Food,Science & Veterinary Medicine, Veterinary Science Centre, Belfield Dublin 4 Biosafety of nanomaterials is a relevant issue both for the industry and medical field as their use ranges from computer industry, orthopaedics and medical applications as diagnostics tools, therapeutic agents and drug delivery vehicles; it is therefore of primary importance to assess, understand and manage their toxicity. Amino‐modified polystyrene nanoparticles (PS‐NH2 NPs) can easily be modified to carry chemicals and proteins, therefore they are an interesting model for drug delivery; Cerium Oxide (CeO2) NPs are used in ceramics, to produce photosensitive glass, and in catalytic converters in automotive applications. Preliminary results from IANH did not reveal toxic effects of CeO2 NPs, while our research has revealed that PS‐NH2 NPs cause apoptotic cell death by inducing cytosolic calcium increase, lysosomal damage, mitochondrial membrane depolarization and activation of the caspase cascade in astrocytoma cells. These two nanoparticles were chosen to establish an HCA platform to assess NPs toxicity and to further characterize apoptosis induced by several classes of nanomaterials. The cell lines chosen were HepG2 as an in vitro model for liver toxicity and 1321N1 cells which are of interest as a model for targeted nanoparticles drug delivery across the blood brain barrier. Immunophenotyping, its role in Acute Leukaemia David O’Brien Haematology Dept., St.James’s Hospital, Dublin The presentation will introduce the use of immunophenotyping by flow cytometry in Acute leukaemia both in Diagnosis and residual disease monitoring. Minimal residual disease detection analysis by flow cytometry compares well with sensitivity levels achievable by molecular techniques and offers a rapid and relatively cheap alternative to PCR. A case history of B Acute Lymphoblastic Leukaemia illustrates the utility of MRD analysis by flow cytometry. Specific immunophenotyping patterns in Acute Leukaemia have been shown to have a strong association with cytogenetic abnormalities. Less well known is the association with molecular defects. Immunophenotype results for a series of Acute Leukaemia cases will be presented with key diagnostic features and with correlations with cytogenetic abnormalities and genetic lesions discussed .
A high throughput flow cytometry assay to screen the cytotoxicity of dual‐drug combinations Egle Passante1, Maximilian Wuerstle1, Markus Rehm1 Department of Physiology and Medical Physics, The Royal College of Surgeons in Ireland, York House, York Street, Dublin 2 Ireland Multi‐drug combinations are widely used tactics to attack multiple cellular targets in highly complex diseases (cancers, infectious and neurological diseases). A well designed drug interaction experiment implies, as a minimum requirement, the screening of cell responsiveness to each agent individually plus their combination at several concentrations. Drugs with an unknown mechanism of action might require concentrations spanning across several orders of magnitude to be screened. By combinatorial explosion, the number of samples increases exponentially with the number of agents and concentrations to be investigated. Due to being laborious and time consuming, traditional tube‐based assays set constraints, and are therefore often restricted to inappropriately small sets of samples. We have developed a high throughput flow cytometry‐based assay to test the cytotoxicity of up to 64 dual‐drug combinations along with their single dose response. The experimental workflow allows for the cultivation, treatment and analysis of both suspension and adherent cells directly in 96 well assay plates. Using stock compound plates for each drug with serially diluted concentrations, we can swiftly create multidrug layouts representing a comprehensive dose combination matrix. Matrix data can be subsequently analysed to assess drug synergy and/or antagonism according to statistical and mathematical algorithms. Our assay offers a quick and inexpensive procedure to analyse a vast range of drug combinations with little manipulation of the samples and addresses one of the most significant bottlenecks in the analysis of multi‐drug (inter)actions and their cytotoxic effects on living cells. To be announced (Current Issues in Lymphoma Diagnosis) Andy Rawstron Leeds Teaching Hospitals, UK Determining the role for Surface Markers of Neutrophil Migration as Predictors of Post‐Operative Clinical Outcome following open heart surgery. S.A. Kennedy1, K. Murphy1, A. Kinsella1,2, Y. Fan1, J.F. McCarthy2, A.E. Wood2, R.W.G. Watson1 : 1: School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin. 2: Department of Cardiothoracic Surgery, Mater Misericordiae University Hospital, Dublin. Introduction Cardiac surgery triggers the immune system, specifically the neutrophil, leading to effects on post‐operative recovery. Our previous work has demonstrated correlations between neutrophil migration rates, tissue damage and post‐operative outcome. Transmembrane proteins such as CD11b, CD47 and CD99 play a key role in the migration of neutrophils from the vasculature to sites of
inflammation. We hypothesise that pre‐operative neutrophil expression of CD47, CD99 and CD11b in response to a stimulus could be used to predict a patient’s outcome following open heart surgery. We also hypothesise than an even more accurate prediction of post‐operative outcome can be achieved by combining our immunological markers of interest (CD11b, CD47, and CD99) with the well established EuroSCORE, which is currently used to predict a 30 day post‐operative mortality following cardiac surgery. Methods A pre‐operative blood sample (20mls) was collected from n=21 patients (following informed consent) undergoing Open Heart Surgery in the Mater Misericordiae University Hospital. Whole blood neutrophil surface CD11b, CD47 and CD99 was determined by Flow Cytometry with or without LPS stimulation. Post‐operative clinical parameters collected included Creatinine levels, hours intubated and ICU stay. Results Increased pre‐ to post‐operative Creatinine levels – a marker of kidney injury, correlated with pre‐operative neutrophil CD11b (p=0.003, r=0.635), and CD47 expression (p=0.0004, r=‐0.740). In addition we demonstrated a significant correlation with the EuroSCORE, ICU stay and pre‐operative renal function (p
BIOPHOTONIC AND SYSTEMS BIOLOGICAL INVESTIGATION OF CELL DEATH SIGNALING Markus Rehm Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland. phone: 00353 (0)1 4028563; email: mrehm@rcsi.ie Intracellular cell death signalling networks comprise dozens of simultaneous variables, amongst them protein concentrations, trafficking rates, and transmembrane potentials. Decades of biochemical research identified, isolated and precisely characterized many protein components of these networks. To understand their biological function, quantitative single‐cell photonics can provide physiologically highly relevant data on intracellular signalling dynamics in time and space. However, only a limited number of cellular parameters can be detected in parallel. To overcome these limitations and to analyse cell death execution on a systems level, we feed both biochemical and imaging data into computational models of cell death execution and can quantitatively predict experimental cellular responses. Here I will (i) demonstrate the main principles of quantitative photonics and systems modelling approaches, (ii) describe how their combination can significantly extend the explanatory power of experimental studies, and (iii) demonstrate how this approach can be employed to inform and guide subsequent research strategies towards identifying suitable treatment paradigms for highly resistant human cancers. New Advances in Flow Cytometry Software John Tigges, Vasilis Toxavidis Harvard University, USA Multicolour flow cytometry analysis in RSV infection Niga Nawroly National Heart and Lung Institute, Imperial College London, UK Respiratory Syncytial Virus (RSV), the major single cause of infantile hospitalization in the Western world. Natural infection causes only weak immunity to re‐infection. There is no vaccine, and experimental vaccines have a history of sometimes enhancing disease severity. The reasons for this are complex but include failure to induce good quality antibody and strong induction of disease‐causing T cells. In experimental models, specific viral proteins and specific T cell subsets have been shown to be associated with distinct types of disease: CD8 (cytotoxic) T cells cause 'shock lung' (and neutrophil efflux into the lung), while Th2 cells may sometimes cause eosinophilic bronchiolitis. Our current focus is on innate immunity (type 1 interferons, NK cells and macrophages), the regulation of immune responses and mechanisms of delayed effects of viral infection. In this talk, the role of flow cytometry will be shown in studying the immunology of RSV infection.
Monitoring Fermentation Performance of Distilling Yeast (Saccharomyces cerevisiae) During Scotch Whisky Fermentation using Flow Cytometry Walker, J. W., Bringhurst, T. A., Brosnan, J. M. and Pearson, S. Y. The Scotch Whisky Research Institute, The Robertson Trust Building, Research Avenue North, Riccarton Campus, Edinburgh, Scotland, EH14 4AP. Fermentation is an important part of Scotch Whisky production that has undergone a number of changes in recent years to increase fermentation efficiency and reduce production costs. As a result, the distilling yeast, Saccharomyces cerevisiae, must be able to tolerate the demands placed upon it during alcohol production. Distilling yeast needs to be of excellent quality, have a very high viability, be able to ferment rapidly to compete with indigenous bacteria present in the process and tolerate numerous fermentation stresses e.g. osmotic shock, increases in temperature, changes in pH and acidity, decrease in substrate availability and increase in ethanol concentration, encountered as fermentation develops. In the past, distilleries have used traditional microbiological techniques to assess yeast quality and monitor fermentation performance but these techniques can be very time consuming and labour intensive. In recent years, at SWRI we have been looking for alternative methods such as flow cytometry, to assess yeast fermentation performance and response to stress more rapidly and efficiently. This study describes research carried out to monitor yeast quality from different suppliers and demonstrates how flow cytometry can be used effectively to assess yeast fermentation performance and stress‐response. We have looked at yeast viability and yeast vitality using a number of fluorescent markers to determine intracellular glycogen, trehalose and neutral lipids in response to changes in process parameters. We have found that the flow cytometer has provided us with important data on yeast quality and fermentation performance and is proving to be an extremely useful research tool which will help to improve our understanding of yeast physiology and metabolism during the Scotch whisky fermentation process. Flow cytometric studies in microalgae Simrat Kaur1, Rhodri Ceredig2, Dagmar Stengel3 and Charles Spillane1 1Genetics and Biotechnology Lab, Botany & Plant Science, School of Natural Sciences, NUI Galway,Ireland; 2National Centre for Biomedical Engineering Science (NCBES), NUI Galway, Ireland; 3 Botany and Plant Science, School of Natural Sciences, NUI Galway, Ireland. Microalgae are photosynthetic microscopic organisms which are ubiquitous in marine, freshwater and terrestrial habitats with a size range of 0.2‐200 µm. Microalgae produce combinations of pigments such as chlorophylls, carotenoids, fucoxanthin, and phycobilins that are responsible for their autofluorescence. Carotenoids are responsible for the primary coloration to microalgae. For example the brown coloration of diatoms is due to the presence of fucoxanthin. The distribution of these light harvesting molecules amongst algal groups can be used as taxonomic classifiers at the division level. Based on their size, shape and pigment composition we have developed flow cytometric procedures to resolve heterogeneous (mixed) populations of microalgae in sampled aquatic samples. The flow cytometric methods we have developed provide a tool for microalgal researchers to perform isolation of pure strains, and facilitates phenotypic screening for ecological and economically important
microalgae (which otherwise is time consuming and laborious task). Our experiments with pure
microalgal cultures have facilitated to optimize a method for an automated isolation of microalgae from
marine water samples. Further experiments on flow cytometer assisted cell sorting of microalgae is
underway to obtain pure cultures from natural waters and also to develop the screening protocols for
cell viability and cellular bio‐molecule content using fluorescent stains.
POSTERS
01 Pneumolysin‐mediated NLRP3 activation is required for protective immunity against
respiratory pneumococcal infection.
Edel A. McNeela1, Cathy Baxter1, Sarah Smeaton2, Rana El‐Rachkidy2, Rachel M. McLoughlin3, Andres
Mori1, Aras Kadioglu2 and Ed C. Lavelle1.
1
Adjuvant Research Group, School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
2
Department of Infection, Immunity and Inflammation, University of Leicester, LE1 9HN, UK. 3School of
Biochemistry and Immunology, Trinity College Dublin, Ireland.
02 Exchange protein directly activated by cAMP and protein kinase A play distinct roles in cAMP‐
mediated effects on innate and adaptive immunity
Eimear Lambe, and Ed C Lavelle
Trinity College Dublin
03 Study of cell death induced by 50 nm amine‐modified polystyrene nanoparticles in 1321N1
astrocytoma cell line
Fengjuan Wang1, Mariana G. Bexiga1,2, Iseult Lynch1, Anna Salvati1, Kenneth A. Dawson1
1
Centre for BioNano Interactions, University College Dublin, Belfield, Dublin 4, Ireland; 2 PhD
Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology,
University of Coimbra, 3004‐517 Coimbra, Portugal
04 MODE OF CELL DEATH IN DIRECTLY IRRADIATED CELLS AND CELLS EXPOSED TO MEDIUM
FROM IRRADIATED CELLSK. Kumar Jella1*, A. Garcia1, B. McClean2, H.J. Byrne3, and F.M. Lyng1 1 Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Kevin St, Dublin 8; 2 St Luke’s Hospital, Highfield Road, Rathgar, Dublin 6; 3 Focas Institute, Dublin Institute of Technology, Kevin St, Dublin 8 05 Whole body flow cytometry: cnidarians show the way Katrin Hensel1, Rhodri Ceredig2 & Uri Frank1 1 School of Natural Sciences & Ryan Institute, NUI Galway; 2 Regenerative Medicine Institute (REMEDI), NUI Galway 06 Rapid Assessment of Growth Performance of Biofuel‐directed Micro‐algae Using Multi‐ parameter Flow Cytometry Liam Brennan, Alfonso Blanco Fernández, Philip Owende University College Dublin 07 Detailed Immunological Profiling of Kidney Injury by Flow Cytometry and Sorting of Rare Cell Populations Shirley Hanley PhD, Jana Pindjakova PhD, Michelle Duffy BSc, Michelle Holland BSc, Rhodri Ceredig MD PhD, Matthew D. Griffin MB BCh DMed Immunology and Transplant Biology Laboratory; Regenerative Medicine Institute (REMEDI); College of Medicine, Nursing and Health Sciences; NUI Galway. 08 Use of flow cytometry to analyse the glycobiology of mouse lymphoid and mesenchymal stromal cell interactions. Shirley Hanley PhD, #Jared Gerlach PhD, *Claas Baustian BSc, #Lokesh Joshi PhD, Rhodri Ceredig MD PhD, Matthew D. Griffin MB BCh DMed Immunology and Transplant Biology Laboratory; Regenerative Medicine Institute (REMEDI); College of Medicine, Nursing and Health Sciences; NUI Galway, #Glycoscience Research Cluster, NUI Galway, *University of Rostock, Germany.
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