Discussion Forum: Flow Cytometry - 4th November 2011

The Penridge Suite, 470 Bowes Road, London, N11 1NL, United Kingdom: Friday 4^th November 2011

This discussion workshop will cover a variety of topics, including advances in instrumentation and techniques, intracellular antigen detection, advances in flow microbiology and advances in the measurement of cell death. We have invited 6 experts to discuss their work in an informal lecture setting, discussion and demonstration groups, one2one sessions and panel discussions.

Meeting Chair: Dr Gary Warnes,Flow Cytometry Core Facility, Blizard Institute of Cell & Molecular Science, Barts and The London School of Medicine and Dentistry, London University

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 Mickey Ramalho, EuroSciCon, London, UK

9:35 – 9:40 Introduction by the Chair: Dr Gary Warnes

Flow Cytometry Core Facility, Blizard Institute of Cell & Molecular Science, Barts and The London School of Medicine and Dentistry, London University

9:40 – 10:00 Cell Death in All its Forms

Dr Gary Warnes

Flow Cytometry Core Facility, Blizard Institute of Cell & Molecular Science, Barts and The London School of Medicine and Dentistry, London University

Cell necrobiology includes the broad range of modes of cell death including classic apoptosis or Type I apoptosis, caspase-independent apoptosis-like programmed cell death (PCD), autophagy or Type II apoptosis, mitotic catastrophe, necrosis-like PCD, oncosis or accidental cell death and cell senescence. The talk will cover most standard flow cytometric techniques currently available to study apoptosis, including, DNA content analysis, use of DNA dyes, annexin V assay, mitochondrial function, caspase activation, Reactive Oxygen Species (ROS) generation, plasma membrane asymmetry dyes, cytochrome c release, Apoptosis Inducing Factor (AIF) translocation measurements. These assays will also be discussed in terms of oncosis in a time-dependent and real-time manner.

10:00 – 10:20 The Optimal Use of Multiple Fluorophores and Dyes in Flow Cytometry

Mr Ian Dimmick

Flow Cytometry Core Facility Manager

Newcastle Upon Tyne University, UK

There are now many fluorophores available, however sometimes the exact excitation and emission is not allways well defined with respect to multiple laser instruments, the user must be capable of determiing the suitability of new fluorophores for their specific flow cytometer .

10:20 –10:40 Advanced Solutions for Flow Cytometric Sorting

Prof Anna Petrunkina

Head of Flow Cytometry

Cambridge Institute for Medical Research, UK

10:40 – 11:00 Mid-morning Break

11:00 – 11:20 Experimental Design in Multi-colour Flow Cytometry

Mrs Niga Nawroly

Flow cytometry consultant

FloWise Scientific

Flow cytometry is a method for studying and analysing expression of cell surface and intracellular molecules. This can be by characterizing and defining different cell types in heterogeneous cell populations. One of the applications of flow Cytometry, is Multi-colour flow analysis, this technique can be used to answer complex cell biological and immunological questions. It reveals more complex information about individual cells at a single cell level, making this technique a very useful tool. But, it comes with some difficult set of technical issues that must be overcome by proper experimental design. In this talk, I will describe the basic aspects of multi-colour flow cytometry, controls, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles.

11:20 – 11:40 Practical considerations for Intracellular Flow Cytometry

Dr Jane Limer

Applications Manager

BioLegend UK Ltd

The success of any flow cytometry assay is dependent on optimised cell preparation techniques. This is especially crucial for intracellular applications, where the choice of fixative and staining protocol can directly influence the staining outcome and data interpretation. Protocol requirements can differ according to antigen location, cell type and fluorochrome choice. Here we discuss the practical considerations for protocol design.

11:40 – 12:00 TBC

Merck Chemicals

12:00 – 12:30 Working Lunch

Please collect your lunch and take it to your discussion table (Session 1)

12:30 – 15:25 Discussion Groups (Sessions 1-7)

• Round table discussion groups (20 minutes each) will be held throughout the afternoon
• Delegates will rotate so that they may participate in all the discussion tables
• All delegates will also be allocated a session for visiting the exhibition stands
• Where appropriate delegates will be able to bring their samples to the discussions
• See end of agenda for description of discussion tables

15:30 – 16:30 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

16:35 Chairman’s Summing Up and Feedback Prize Draw

Round-table Discussion Sessions:

Table A: Cell Death in All its Forms

Hosted by Dr Gary Warnes, whose interest in flow cytometry started at St. Mary’s in 1986, analysing T-cell subsets. Then set up a new flow cytometric T-cell subset service at St.Thomas’ Hospital. Completed a PhD investigating the immunosuppression of HIV-ve haemophiliacs at St.Thomas’ Hospital. Post-doctoral position, investigated the regulation of Tissue Factor expression by immune costimulatory molecules in sepsis. Then managed the Flow & Imaging Core Facilities at the MRC Clinical Science Centre at Hammersmith Hospital. Worked with Derek Davies at Cancer Research UK. Currently managing the Flow facility at the Blizard Institute of Cell & Molecular Science, Queens Mary’s College

Table B: Multicolour Flow Cytometry

Hosted by Mr Ian Dimmick, who worked within Immunology and Haematology clinical environment , moved on to a flow cytometry applications and product specialist role , then into academia at Newcastle University running a busy cell sorting and anlysis laboratory covering a very wide range of applications .

Table C: Troubleshooting Sorting Experiments and Developing Practical Solutions for Successful Sorting

Hosted by Dr Anna Petrunkina, who is Head of Flow Cytometry at the Cambridge Institute for Medical Research and Associate Professor at the University of Veterinary Medicine Hannover. After research period in theoretical physics in St-Petersburg she switched fields and conducted research in reproductive biology and biomedical engineering in Germany. Her research focused primarily on understanding the mechanisms that regulate sperm function. In 1997 she started to use flow cytometry to identify factors affecting fertility. During this time, Anna has set up a departmental cytometric facility and became increasingly fascinated by this technology. In 2007, she took up her present position in Cambridge to direct flow cytometric services. Anna is an Associate Editor of Reproduction, Fertility, Development and retains active research interests in reproduction and cryobiology.

Table D: Intracellular flow cytometry applications

Hosted by Dr Jane Limer, whose background is in academia, working in cancer biology and stem cell research. Then she moved on to a commercial environment, working a flow cytometry training and applications-based roles. Experience in a wide variety of flow cytometry applications.

Table E: Troubleshooting in Multi-colour Flow

Hosted by Mrs Niga Nawroly, who is an immunologist, and was Head of flow Cytometry in Imperial College for 10 years. Now, a consultant in FloWise Scientifics and Business Development manager in eBioscience.

Keywords:

Multi-colour flow cytometry, experimental controls, compensation, intracellular antigens, DNA, cytokines, cell sorting, instrumentation, design, preparation, viability, cryopreservation

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