BD FACSamples and Recommendations

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1 Eesti FACSi Kasutajate Konverents Advances in Flow Cytometry Tallinn Meriton Grand Conference & Spa Hotel

2 Eesti FACSi Kasutajate Konverents Advances in Flow Cytometry Tallinn Edited by Janne Pullat, PhD BOOK OF ABSTRACTS 2

3 PRESENTATIONS: The history and future of the BD Accuri Flow Cytometers Håkan Samuelsson, PhD BD Accuri, Nordic & Baltic Field Application Specialist The mini flow cytometer Accuri C6 with and origin in Michigan Uninversity has become popular all over the world since its release 5-6 years ago. The secret is that the Accuri instrument and the software were developed hand in hand with researchers and they did not rush to put it on the market just to get quick money. Since the realease it has continously been development, hand in hand with reaserchers, for keeping it a very strong alternative technology. Why did BD buy Accuri and nothing else? It was a very hard competitor in the small class with a Blue and a Red laser, FL1-4 and FSC/SSC. Since the transfer 2011, BD has developed the standard BD Accuri instrument and also new 1 or 2 laser BD Accuri instruments, just released, with Blue, Red, UV, Violet, Lightgreen, YellowGreen laser options...and future developments will come perhaps in collaboration with you. 3

4 Compensation in Multicolor flow -Tips & Tricks Pernilla Eliasson, PhD BD Biosciences Application Specialist Over the past decade the number of colors analyzed in typical flow cytometry experiments has increased and in many laboratory facilities today 8 and more colors panels are routinely and frequently used. This is enabled due to high performance instrumentations with additional lasers and detectors options as well as development of more fluorochrome options thanks to advanced biochemical research progressions. In order to perform accurate analysis compensation is essential in the setup for multicolor assays. Compensation can for many flow cytometry users feel complicated and problematic. In this presentation basics of spillover from colors and tips for setting up a correct compensation will be discussed. "Multicolor Flow Cytometry How to optimize a multicolor panel and get reproducible results" Sara Johansson, PhD BD Biosciences Scientific Support This presentation will focus on multicolor flow cytometry and give an update about unique tools for instrument quality control and standardization. The aim is to understand how to choose the best possible combinations of fluorochromes and how to improve the reproducibility of multicolor flow cytometry data. It will also highlight the characteristics of both classical and new very bright fluorochromes. Experimental data will be used to illustrate how to build and optimize a multicolor staining panel with a focus on do s and don ts. 4

5 New Products from BD Biosciences Anna Cannava, PhD BD Biosciences Product manager Cellular Analysis Cell sorting The BD FACSAria III where innovation comes as standard and introducing the new BD FACSJazz Morgan Blaylock, PhD BD Biosciences Innovation has always been built into the BD FACSAria. The new generation BDFACS Aria III represents the latest innovations, offering advances that deliver reproducible results and superior performance. Since the introduction of the first BD FACSAria in 2003, each successive generation has opened the complex world of high end cell sorting to a broader audience of researchers and wider range of applications. The current BD FACSAria has innovations which provide superior multicolour performance offering you a system which is expandable for the future. The BD FACSAria III-in a perpetual state of the art to support your next great discovery. The BD FACS Jazz is the newest in our line of sorters it signals a new era in cell sorting with dependable BD performance, but with benchtop fit and an affordable price. The BD FACSJazz cell sorter incorporates design features that simplify operation of stream-in-air cell sorters. The system offers factory-optimised settings, intuitive alignment, real time video monitoring and BD FACS Accudrop technology. The BD FACSJazz has many innovations that make it a powerful, approachable tool for experienced and novice flow cytometry users alike. 5

6 Flow Cytometry analysis in the Core Facility of Institute of Technology, University of Tartu Reet Kurg, PhD Institute of Technology, University of Tartu 1000 and 1 way to use flow cytometer Dmitri Lubenets, MSc University of Tartu, Faculty of Science and Technology I will speak about different flow cytometric assays involving bacteria, yeast, small exosomes, micro vesicles, stem cells and describe them from the point of view of flow cytometry specialist. 6

7 Optical filters in flow cytometry Michael Sommerauer, PhD AHF analysentechnik AG, Tübingen, Germany Optical filters are essential tools for fluorescence detection in microscopy as well as in flow cytometry. Major improvements in coating technologies increased the performance of optical filters significantly. This talk will answer the question which filters usable for fluorescence detection followed by a comparison of different filter types. A short introduction into coating technologies will finalize the first part of this lecture. The second part show the use of optical filters in flow cytometers and the requirements they have to fulfill. Different types of detection setups will be presented including their advantages and drawbacks from an optical filter point of view. Practical Experience with Optical Filters in Flow Cytometry Joachim Brenner, PhD, BD Biosciences, European Technology Center (ETC), Heidelberg, Germany In multi-laser and multi-color instruments it is getting more and more important to choose correct filters with the best optical specifications. BD Biosciences delivers its instruments with bandpass filters and dichroic mirrors for a broad range of applications. Nevertheless there is no single best filter combination for all instruments with different lasers and applications available. In some cases, especially with customized instruments (SORP), it might become necessary that customers have to choose appropriate filters, and order it directly from optical companies or distributors. Therefore I will give some recommendations for choosing the right filters. I will first talk about bandwidth and spectra vs blocking of filters, in order to achieve the best performance (high StainIndex) with a certain fluorochrome and laser combination. 7

8 Examples will be given for PE and APC, where different filters and their results are compared depending on the choice of lasers. Furthermore I will talk about NOTCH filters and their advantages and disadvantages. Finally I will talk about laser power and sensitivity (separation/stainindex) according to different fluorochromes and autofluorescence of cells and beads. Efficient and durable linear variable filters and dichroics Oliver Pust, PhD Business Development Manager Optical Thin Film Filters at Delta Dynamics of Bacterioplankton in the Gulf of Finland Peeter Laas, MSc Tallinn University of Technology, Institute of Marine Systems The Gulf of Finland is one of the most eutrophied parts of the Baltic Sea. The mean water circulation in the Gulf of Finland is cyclonic, the inflow of the saltier northern Baltic Proper water occurs along the Estonian coast and the outflow of the fresher Gulf water (influence of River Neva) occurs along the Finnish coast. The general cyclonic water circulation might have a considerable effect to the differences between two coastal areas resulting e.g. the variation of composition and abundance of microbial communities in different parts of the Gulf. Vertical profiles of different environmental variables along the North-South cross-section in the central part of the Gulf were measured aboard RV Salme (Marine Systems Institute at the Tallinn University of Technology) in 2011 and During each, cruise sampling at 6 stations (various depths) was conducted. In total 140 samples collected throughout ice-free period from fall 2011 to fall 2012 were included in the present analysis. The samples were run on BD LSR II 8

9 Flow Cytometer at the Institute of Technology at University of Tartu. Our goal was to study spatial and temporal dynamics of pikoplankton abundance, size and trophic state (heterotrophic/autotrophic). Our long-term goal is to make this kind of studies routine in the Gulf of Finland area. Flow Cytometry for Clinical Applications Olga Aavasalu, MSc North Estonia Medical Centre The presentation will focus on flow cytometry for clinical applications. In the first part of this presentation I will give an overview of antibody panels needed in diagnostics of leukemia, lymphoma, myeloma etc. The talk will also include the changes in monoclonal antibody panels used in diagnostics of ALL and AML in NOPHO project. In the second part of the presentation I will give an overview of the study made in The North Estonia Medical Centre there we compared neoplastic and normal phenotypes of plasma cells to distinguish multiple myeloma and reactive atypical plasmacytosis cases, of plasma cells to distinguish multiple myeloma and reactive atypical plasmacytosis cases, and compose the most useful panel of monoclonal antibodies for multiple myeloma diagnostics using flow cytometry immunophenotyping method. Signal transduction via STATs: getting insight into disease mechanisms Kai Kisand, MD, PhD University of Tartu 9

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11 Characterization of human natural killer cell culture by flow cytometry K.Värv, R. Rumvolt and M.Drews Competence Centre for Cancer Research and University of Technology, Akadeemia tee 15, 12618, Tallinn, Estonia Natural killer (NK) cells are lymphocytes of the innate immune system that have the ability to recognize and lyse abnormal body cells without prior stimulation. As these cells play an important role in the immunosurveillance of malignant diseases, in vitro expansion of human NK cells from peripheral blood mononuclear cells (PBMC) is a growing perspective tool in cellbased cancer therapy. In current work the optimization of cultivation strategies is based on growth dynamics, viability, total amount of viable cells and percentage of NK cells in culture. NK cells derived from three different donors were expanded on CellGro medium with bioactive supplements by continuous monitoring of human natural killer specific cell surface markers (NKp46, NKp44, CD3 and CD56) using flow cytometry. From PBMC of 50 ml of donor blood we obtained culture with absolute number of expanded viable cells up to 1,2*10 9 by the 25 th day of cultivation. Maximal mean percentage of NK cells in culture was determined by NKp46 cell surface marker between cultivation days of and by NKp44 marker between days of Monitoring of cultivation efficiency by cell growth parameters and surface markers with flow cytometry analysis is essential prerequisite for high quality outcome in in vitro expansion of NK cells. 11

12 Results of current work show that in vitro expansion of human NK cells on CellGro medium with different GMP-compliant bioactive supplements is a usable method for application in medical use. 12