Techniques for Immune Function Analysis - Office …

1 BD BiosciencesTechniques for ImmuneFunction AnalysisApplication Handbook 1stEditionFor Research Use Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include orcarry any right to resell or transfer this product either as a stand-alone product or as a componentof another product. Any use of this product other than the permitted use without the express writtenauthorization of Becton Dickinson and Company is strictly applications are either tested in-house or reported in the literature. See Technical Data Sheets for , BD Logo and all other trademarks are the property of Becton, Dickinson and Company. 2003 BDFor additional informationplease access theImmune Function Homepage of ContentsPreface.

2 4 Chapter 1: Immunofluorescent Staining of Cell Surface Moleculesfor Flow cytometric Analysis .. 9 Chapter 2: BD cytometric Bead array (CBA) Multiplexing Assays .. 35 Chapter 3: BD DimerX MHC:Ig Proteins for the Analysis ofAntigen-specific T Cells.. 51 Chapter 4: Immunofluorescent Staining of Intracellular Moleculesfor Flow cytometric Analysis .. 61 Chapter 5: BD FastImmune Cytokine Flow Cytometry .. 85 Chapter 6: BD ELISPOT Assays for Cells That SecreteBiological Response Modifiers .. 109 Chapter 7: ELISA for Specifically Measuring the Levels of Cytokines,Chemokines, Inflammatory Mediators and their Receptors .. 125 Chapter 8: BD OptEIA ELISA Sets and Kits for Quantitationof Analytes in Serum, Plasma, and Cell Culture Supernatants.

3 143 Chapter 9: BrdU Staining and Multiparameter Flow cytometric Analysisof the Cell Cycle .. 155 Chapter 10: Cell-based Assays for Biological Response Modifiers .. 177 Chapter 11: BD RiboQuant Multi-Probe RNaseProtection Assay System .. 197 Chapter 12: Tools to Study the Complement System .. 229 Chapter 13: Detection of In VivoCytokine Productionwith the In VivoCapture Assays for Cytokines .. 243 Acknowledgements .. 246 Unless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for the CoverA set of graphics was selected that represent the various technologies,applications, and reagents offered by BD Biosciences, which are useful forstudying Immune Function .

4 The graphics were mapped together onto the surfaceof a sphere, which symbolizes a cell, the fundamental biological unit involvedin the generation and mediation of immunological and inflammatory composite graphic represents the integrated set of tools and solutions thatare available for multiparameter, high-resolution analyses of the molecular andcellular mechanisms that underlie Immune study of the Immune system attracts large numbers of researchers fromdiverse scientific disciplines because of its central importance in providingimmunological host defense and its intercommunication with other systems thatmaintain bodily homeostasis. The Immune system is often studied in its intactform but it can also be readily disassembled into its cellular and molecularcomponents (eg, lymphoid cell populations and effector molecules), recombinedand modified in various ways, and analyzed in an in vitroor an in to the creative development and application of a wide variety ofexperimental protocols, often using new technological platforms and reagents,vast amounts of new information concerning Immune Function become availableon a daily basis.

5 Researchers busily scrutinize this information hoping to betterdefine and understand the networks of cellular and molecular mechanisms thatunderlie immunity and inflammation in health and Biosciences is pleased to introduce the new Techniques for Immune FunctionAnalysis, Application Handbook 1stEdition. This handbook grew out of theoriginal Cytokine/ Chemokine Application Manual that was first published in original manual was based on BD Biosciences technical publications andpresentations and with a tremendous amount of input from customers dealingwith Immune Function studies from a Genes to Proteins to Cells new title for this publication reflects the enlarged scope of the book thatserved as a guide for applications and reagents designed to study the roles playedby cells and the regulatory and effector molecules (ie, biological responsemodifiers including cytokines, chemokines, inflammatory mediators and theirreceptors) that mediate inflammation and natural and acquired immunity.

6 Newchapters dealing with the BD cytometric Bead array , the BD FastImmune System, the BD ELISPOT Assay, BD DimerX MHC:Ig Molecules,Immunofluorescent Staining of Cell Surfaces for Flow cytometric Analysis , andInflammatory Mediators have been added to this handbook. Previous chapterspertaining to the BD RiboQuant Multi-Probe RNase Protection Assay System,ELISA, BD OptEIA ELISA Sets and Kits, Immunofluorescent Staining ofIntracellular Molecules for Flow cytometric Analysis , and Bioassays have beenrevised as well with a presentation of new reagents and methods discussedtherein. For additional information please access the new Immune FunctionHomepage at otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures.

7 Not for , 2 -Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) AEC3-amino-9-ethyl-carbazoleakaalso known asAPCallophycocyanin or antigen-presenting cellBrdUbromodeoxyuridineBRMbiological response modifierBSAbovine serum albuminCO2carbon dioxidecpmcounts per minuteDAPI4 ,6-diamidino-2-phenylindole*2 HClddH2 Odistilled deionized waterDMFdimethyl formamideDMSO dimethyl sulfoxideDTTdithiothreitolED5050% effective doseEDTA ethylenediamine tetraacetic acidELISA enzyme-linked immunosorbent assayELISPOT enzyme-linked immunospot assayFACS fluorescent activated cell sortingFcRimmunoglobulin Fc receptorsFBSfetal bovine serumFITC fluorescein isothiocyanateGM-CSFgranulocyte-macropha ge colony-stimulating factor H2O2hydrogen peroxidehrhourHRPhorseradish peroxidaseIFNinterferonIgimmunoglobulinI LinterleukinkDakilodaltonLliterLALlimulu s amebocyte lysateLPSlipopolysaccharideMCPmonocyte chemoattractant proteinminminuteMIPmacrophage inhibitory proteinmRNAmessenger RNANA/LEno azide/low

8 EndotoxinND5050% neutralizing doseODoptical densityNBCS newborn calf serumPAGE polyacrylamide gel electrophoresisPBSphosphate buffered salinePBS-TweenPBS containing Tween-20 PEphycoerythrinPerCPPeridinin chlorophyll proteinpfuplaque forming unitsPBMC peripheral blood mononuclear cellsPIpropidium iodideUnless otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for (continued)PMAphorbol myristate acetatePMTphotomultiplier tubePYpyronin YRANTESR egulated upon Activation, Normal T Expressed and presumably SecretedrhILrecombinant human interleukinRNAribonucleic acidRPAribonuclease protection assayRTroom temperatureSDSsodium dodecyl sulfate[3H]-TdRtritiated thymidineTBETris borate EDTATCC terminal complement complexTCRT cell receptorTDST echnical Data SheetsTETris EDTATMB tetramethylbenzidineTNFtumor necrosis factorUunit1.

9 BD Cy-Chrome is now listed as TNF- is now listed as otherwise specified, all products are for Research Use for use in diagnostic or therapeutic procedures. Not for 1 Immunofluorescent Staining of Cell SurfaceMolecules for Flow cytometric Analysis ofImmune FunctionIntroductionTo understand Immune responses, it is necessary to identify, isolate, and studya variety of cell types, cell functions , and interactions that constitute thoseresponses. A vast array of different cell surface molecules are involved inmediating Immune responses. Methods that determine the types and levelsof such membrane molecules (surface markers) that are co-expressed by cellsprovide important information regarding cell lineage, activation status, adhesion,migration and homing capacity, and ability to respond to stimuli and to interactwith other cells.

10 For the purposes of this handbook, this chapter will focus onmethods for the detection and measurement of cell surface molecules thatmediate cellular functions by virtue of their expression and/or binding ofsignaling molecules that are critical for cellular intercommunication. Suchsignaling molecules include cytokines, chemokines, inflammatory mediators,and their receptors, (ie, biological response modifiers [BRMs] of the ImmuneSystem). Upon interaction with their specific receptors, BRM ligands caninfluence the physiology of either the producer cell (autocrine action), adjacenttarget cells (paracrine action) or distant target cells (endocrine action).In this way, BRMs may influence target cell activation, growth, proliferation,differentiation, migration, and effector Function (eg, expression of other BRMs).