An event from European Scientific Conferences - Euroscicon "Specialising in communicating cutting edge technology & methodology in the Life Sciences"
This event is a discussion forum, focused on comparing and contrasting available techniques that measure cell mediated immunity. The study of cell mediated immunity requires the identification of cell types and subsets involved, accurate determination of levels of effector molecules, and the kinetic assessment of these same effector molecules in real time. Advances in Fluorescence Activated Cell Sorting (FACS) allow the assessment of multiple effector molecules, via intracellular antigen detection, in defined cell subsets, via extracellular antigen detection. However, it does not allow the precise and timely assessment of these same factors, especially after re-stimulation of cell samples. Enzyme-Linked ImmunoSorbent Assay ELISA and Cytometric Bead Array (CBA) allow the accurate determination of one or multiple effector molecules, but information on their cellular origin is often sacrificed. Participants will have a chance to explore the advantages and pitfalls of effector molecule detection with the experts during round table and panel discussions
Meeting Chair: Dr Marc Veldhoen, The Babraham Institute, Cambridge, UK
This event has CPD accreditation
On registration please submit your questions to the panel that will be asked by the chair on the day of the event
9:00 – 9:30 Registration
9:30 – 9:35 Introduction by Meeting Coordinator: Dr Astrid Englezou, EuroSciCon, London, UK
9:35 – 9:45 Introduction by the Chair: Dr Marc Veldhoen, The Babraham Institute, Cambridge, UK
Talks by Invited Experts:
9:45 – 10:15 Flow cytometry and cytokine detection
Dr Marc Veldhoen
Babraham Institute, Cambridge, UK
10:15 – 10:45 Use of Multiplex Cytokine Analysis in Determining Secretion Profiles of Leucocyte Populations in the Female Reproductive Tract
Dr Gendie Lash
Lecturer at Newcastle University
Leucocytes are a major cell population in the non-pregnant endometrium and early pregnancy decidua. Some of these cells have non-immune functions in addition to their classical immune functions. To decipher the functions of two of these cell types, uterine macrophages and uterine natural killer (uNK) cells we have used multiplex cytokine and growth factor analysis to determine the secretion profiles of these cell types. This has led to hypothesis driven functional studies based on their secretion profiles. Data will be presented on initial studies into choice of multiplex analysis system and the type of data we are able to generate using this approach compared to more conventional ELISA.
10:45 – 11:15 Mid-morning Break
11:15 – 11:45 Fluorescently labeled tetrameric MHC-peptide complexes
Dr Stuart Sims, Nuffield Department of Medicine and NIHR Biomedical Research Centre University of Oxford Oxford UK
The development of the fluorescently labeled tetrameric MHC-peptide complex has enabled the direct visualisation, quantification and phenotypic characterisation of antigen-specific T cells using flow cytometry and has transformed our understanding of cellular immune responses. The combination of this technology with functional assays provides many new insights into these cells, allowing investigation into their lifecycle, manner of death and effector function. This talk will provide an overview of the techniques used in the construction of these tetramers, the problems and solutions associated with them, and the methods used in the study of antigen-specific T cells. Understanding how the antigen-specific cells develop and function in different circumstances and with different pathogens will be key to understanding natural host defence, as well as vaccine design and assessment.
11:45 – 12:15 The use of flow cytometry to dissect vaccine and pathogen induced T cells responses
Dr Phil Hogarth, TB Immunology, Animal Health & Veterinary Laboratory Agency¸UK
Using a murine model of BCG vaccination & M. bovis challenge, we are investigating the T cell responses responsible for providing (driving?) protective immunity. Traditional techniques such as ELISPOT & ELISA combined provide valuable data, but fail to identify the phenotype or multiple functionality of responder T cells. Using Intracellular Staining (ICS) techniques, we are able to generate data describing 7 separate subsets of T cells based on cytokine functionality combined with surface phenotype.
We demonstrate that a single systemic BCG vaccination induces distinct systemic and mucosal populations of T effector memory (TEM) cells in vaccinated mice. These CD4+CD44hiCD62LloCD27- T cells are maintained for long periods in BCG protected mice, maintaining a vaccine–specific functionality.
12:15 – 12:45 FluoroSpot analysis of TLR-activated monocytes reveals several distinct cytokine secreting subpopulations
Christian Smedman, Mabtech AB, Sweden
A hallmark property of monocytes is their ability to rapidly produce and secrete a broad range of cytokines in response to TLR-stimulation. Earlier investigations of this diversity have for the most part been based on methods such as ELISA or Luminex, making it difficult to draw conclusions as to their functional profile on the cellular level. Mabtech has developed a range of FluoroSpot reagents which allows detection and quantification of cells secreting two cytokines simultaneously. By this technique it is possible to identify several subpopulations of monocytes with distinct cytokine secreting profiles in response to ligands against TLR2, TLR4 and TLR7/8. This talk will describe the FluoroSpot methodology which offers a sensitive and robust method for detecting dual cytokine secretion at the single cell level. Possible implications of the results to the general field of monocyte and macrophage biology will also be discussed
12: 45 – 13:15 Working Lunch
Please collect your lunch and take it to your discussion table
This is also a good time to fill out your feedback forms
13:15 – 14:45 Discussion Group Sessions
14:45 – 15:15 Afternoon Tea
15:15 – 16:15 Question and Answer Session
This session will include summing up of the discussion tables and questions submitted both prior to the meeting and throughout the day
16:15 Chairman’s Summing Up and Feedback Prize Draw
About the Speakers
Phil Hogarth started his research career at the Liverpool School of Tropical Medicine, with a PhD in Immunoparasitology. Following postdoctoral training at Bristol University he joined AHVLA (formerly VLA) in 2001, becoming a team Leader/Senior Scientist. His main research interests are the T cell mechanisms by which vaccination protect against TB, and the identification of correlates of vaccine induced protection. Phil’s expertise lies in the use of flow cytometry to identify the phenotype and function of T cells induced by vaccination or infection with TB. He is reviewer for many journals & funding agencies, including the EU and Wellcome Trust and the author of 23 papers.
Stuart Sims is a Wellcome Trust funded research assistant now enrolled for a DPhil. He is studying the transcriptional regulation of memory inflation and CD161 expression.
Gendie Lash, who obtained her undergraduate and PhD degrees in Biochemistry from University of Otago, Dunedin, New Zealand in 1997. She then did Post-Doc jobs in Obstetrics and Gynaecology, University of Nottingham and Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada where she held a Canadian Hypertension Society Post-Doctoral Fellowship. In 2002 she moved to Newcastle University where she has been ever since and is currently a lecturer. Her current research focuses on blood vessel development in the non-pregnant uterus and maternal adaptations to pregnancy, with particular interest in the functional role of uterine leucocytes in these processes.
Keywords: Flow Cytometry, ICS, ELISPOT, BCG, TB, BCG, uNKCytokine, ELISA, Multiplex. TLR. Toll, TB, TLR2, TLR4, FluoroSpot
Meeting Web Site: www.regonline.co.uk/CMIdiscussion2012
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