Techniques for Immune Function Analysis - …

1 Techniques for Immune Function AnalysisApplication Handbook 2nd EditionXEUR 1225-00BD, BD Logo and all other trademarks are the property of becton , dickinson and Company. 2009 BDBD BiosciencesEuropeErembodegem-Dorp 86B-9320 ErembodegemBelgiumTel.: (32) 2 400 98 95 Fax: (32) 2 401 70 BiosciencesWorldwideCell Analysis 2350 Qume DriveSan Jose, CA 95131-1807 USATel.: : : (43) 1 : (43) 1 (32) 53 : (32) 53 (45) 43 43 45 66 Fax: (45) 43 43 41 AFRICATel.: (254) 20 2738339/40 Fax: (254) 20 EUROPETel.: (49) 6221 305 113 Fax: (49) 6221 305 (358) 9 8870 7832 Fax: (358) 9 8870 (33) 04 76 68 37 35 Fax: (33) 04 76 68 35 (49) : (49) (30) 210 : (30) 210 : (36) 1 345 7090 Fax: (36) 1 345 (39) 02 : (39) 02 EASTTel.: (971) 4 : (971) 4 (47) 73591200 Fax: (47) (48) 22 : (48) 22 stica e Farmac utica, LdaTel.: (351) 21 : (351) 21 AFRICATel.: (27) 11 : (27) 11 (34) 90 : (34) 91 (46) 8 775 51 10 Fax: (46) 8 645 08 (41) 61 : (41) 61 NETHERLANDSTel.: (31) 20 : (31) 20 (90).

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3 : (41) 61 485 22 95 Fax: (41) 61 485 22 SupportTel.: (41) 44 580 43 73 Fax: (41) 44 580 43 NETHERLANDSTel.: (31) 10 711 4800 Fax: (31) 10 711 +44 (0) 207 075 3226 Fax: +44 (0) 207 075 Scientific Support for technical product information, product application for Immune Function AnalysisApplication Handbook 2nd EditionAlexa Fluor is a registered trademark of Molecular Probes, Inc. Cy is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855-1327, Array is a trademark of Softflow, is a trademark of GE HealthcareImmunoSpot is a registered trademark of Celluar Technology Limited, Cleveland Ohio. The BD Pharmingen Cytokine ELISPOT Kit and protocols were developed in collaboration with Cellular Technology Limited, Cleveland, is a trademark of NycomedTween is a registered trademark of ICI Americas IncBD flow cytometers are Class I (1) laser products.

4 2009 becton , dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD research use only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of becton dickinson and Company is strictly prohibited. Product availability and prices are subject to change without , BD Logo and all other trademarks are property of becton , dickinson and Company. 2009 BDFor additional information and updated lists of related products, please visit you know about our protocols on the web?

5 Biosciences Scientific SupportOur team of experts is trained to help you with your technical questions about our products and their Tel.: (43) 1 928 04 65 Belgium Tel.: (32) 2 401 7093 Denmark Tel.: (45) 80 882 193 Eastern Europe, Tel.: (32) 53 720 740 Middle East & AfricaFinland Tel.: (358) 800 11 63 17 France Tel.: (33) 1 70 70 81 93 Germany Tel.: (49) 69 222 22 560 Greece Tel.: (30) 0 800 12 7506 Hungary Tel.: (36) 6 800 16245 Ireland Tel.: (44) 207 075 3226 Isra l Tel.: (972) 1 80 921 6283 Italy Tel.: (39) 02 360 036 85 Norway Tel.: (47) 800 18530 Portugal Tel.: (351) 8008 15176 South Africa Tel.: (27) 80 09 81008 Spain Tel.: (34) 91 414 6250 Sweden Tel.: (46) 8 5069 2154 Switzerland Tel.: (41) 44 580 43 73 The Netherlands Tel.: (31) 10 711 4800UK Tel.: (44) 207 075 3226 Table of ContentsPreface ..4 Abbreviations ..7 Chapter 1 Immunofluorescent Staining of Cell Surface Molecules for Flow Cytometric Analysis of Immune Function ..11 Chapter 2 BD Cytometric Bead Array (CBA) for Multiplexed Quantitation of Biological Response Modifiers.

6 31 BD CBA Flex Sets ..34 BD CBA Kits ..61 BD CBA Functional Beads ..67 Chapter 3 Intracellular Cytokine Detection by Flow Cytometry: BD Cytofix/Cytoperm Method ..85 Chapter 4 Intracellular Cytokine Detection by Flow Cytometry: BD FastImmune System 109 Chapter 5 Immunofluorescent Staining of Intracellular Molecules for Flow Cytometric Analysis : Immune Effectors and Signaling Molecules ..131 Chapter 6 BD ELISPOT Assays for Cells That Secrete Biological Response Modifiers ..141 Chapter 7 Cytotoxicity and Degranulation Assays ..155 Chapter 8 Cell Cycle Analysis and BrdU-Based Proliferation Assays for Flow Cytometry, Image Analysis and In Situ Applications ..169 Chapter 9 BD ELISA Reagents for Quantitation of Soluble Biological Response Modifiers ..193 Chapter 10 In Vivo Capture Assays for Sensitive and Direct Measurement of Mouse Cytokine Production In Vivo ..209 Chapter 11 BD Dimer X MHC:Ig Fusion Proteins for the Analysis of Antigen-specific T Cells.

7 219 Chapter 12 Cell-based Assays for Biological Response Modifiers ..231 Chapter 13 Tools to Study the Complement System ..249 Unless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for Function Technologies to Meet Your Experimental NeedsThe Immune system is a focus of study for many researchers from diverse scientific disciplines because of its central importance in host defense and its intercommunication with other systems that maintain bodily homeostasis. Having reliable tools and assay systems that can give you the richest information in the least time is critical to all areas of immunology research, whether your focus is on better understanding the cellular and molecular basis of immunological and inflammatory responses, on measuring immunological endpoints in health and disease, or on developing drugs to modulate the Immune to providing researchers with flexible and comprehensive solutions for exploring Immune Function , BD Biosciences offers a comprehensive range of reagents and assay systems for the functional Analysis of Immune modulators and Immune cell populations.

8 Covering the main Techniques currently in the immunologist s toolbox , we continue to commercialize state-of-the-art technical advances around cell Analysis , helping you to harness the power of flow cytometry for multiplexable, information-rich Immune Function studies. Understanding the importance of assay standardization and increased throughput in clinical research settings, we incorporate these aspects into our assay and application development, to help you keep up with the demands in clinical research technology of choice for your particular experimental needs depends on a number of factors, such as the information needed, sample constraints, and instrumentation available. An overview of the features of each type of assay is shown in Table 1 (page 6), which aims to provide direction in the choice of the appropriate You Keep the PaceAt BD Biosciences we understand that keeping up with technological and methodological advances is key in a laboratory setting. As part of our ongoing commitment to offer you the best service and assistance, we provide you with detailed, carefully optimized protocols for the use of our products.

9 The Techniques for Immune Function Analysis : Application Handbook 2nd Edition is a compilation of protocols and technique -oriented tips and tricks that aim to help you get the most out of your Immune Function original version, published in 1997, included a tremendous amount of input from our customers. Over the years, the collection of protocols and methodological tips has evolved. New additions in this edition include: Expanded chapter on BD Cytometric Bead Array now including the newer create-your-own array protocols New chapter on Cytotoxicity and Degranulation AssaysUnless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for providing you with optimized protocols, we include training and support as an integral part of our offering. Our training courses offer the highest quality education in cell Analysis using state-of-the-art flow cytometry equipment and methodologies. BD Instructors and Flow Support Specialists blend experience in training with technical expertise to offer you the learning you need to stay current with and take full advantage of this changing technology: from hands-on courses in one of the BD Regional Training Centers to personalized assistance with your instruments and applications, to online learning Scientific Support team offers personalized phone and e-mail assistance on our products and their applications.

10 All highly qualified Life Scientists, with in-depth understanding of our products and technologies, they are there to provide you expert assistance on-demand. You may contact them at the numbers on the inside Function Solutions from BD BiosciencesImmune Function Mini-Website: otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for ELISPOT TechnologyDegranulation Assayssuper-antigenTMHCTCRAPCBD DimerXAntigen PresentationBD CytometricBead ArrayBD ELISA ReagentsIntracellularCytokine Detection SystemsBrdU Proliferation AssaysIn VivoCapture Cytokine otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for 1. Tools and Technologies for Immune Function MonitoringTool/TechnologyFlow cytometry - SurfaceFlow cytometry - IntracellularELISPOTMHC mimicsELISAIn Vivo CaptureMultiple Bead ArraysMolecules detectedSurfaceIntracellular & surfaceSecreted (in-situ)SurfaceSecretedSecretedSecreted or intracellularMultiparameter?