DAKO Flow Cytometry Educational Guide

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A nice primer for FACS or Flow cytometry of cells in molecular biology; Fluroscent Activated Cell Sorting

Text of DAKO Flow Cytometry Educational Guide


&,'*&*Educational Guide 2nd EditionF LOW CYTOMET RY08065 15DEC05DonmarkCorporato HoadquartorsTol +45 44 85 95 00Unitod Statos o AmorioaTol 800 235 5743www.dako.comDistributors in morotnan 50 oountriosGuide to FIow CytometryEDITORSonja WulffDako Fort Collins, Colorado, USATECHNICAL ADVISORSKathleen MartinDako Fort Collins, Colorado, USAAngela VandergawDako Fort Collins, Colorado, USACONTRIBUTORSThomas Boenisch, MSDako Carpintoria, Caliornia, USAan Brotherick, PhDDako Ely, Unitod KingdomTerry Hoy, PhD, FRCPathUnivorsity o Walos Collogo o Modioino Cardi, Walos, UKJim Hudson, PhD Dako Carpintoria, Caliornia, USAChristina Jespersgaard, PhDDako Glostrup, DonmarkPeter LopezAaron Diamond ADS Posoaron Contor Now York City, Now York, USAAlberto Orfao, MD, PhDContro do nvostigaoion dol Canoor Salamanoa, SpainW. Roy Overton, PhDDako Fort Collins, Colorado, USAStephen W. PursleyDako Fort Collins, Colorado, USAJohn Ransom, PhDNovasito Pnarmaooutioals San Diogo, Caliornia, USAJohn Sharpe, PhDDoxool Limitod Hamilton, Now ZoalandCarleton C. StewartPoswoll Park Canoor Contor Bualo, Now York, USAan StorieDako Glostrup, DonmarkLars Winther, PhDDako Glostrup, Donmark Ccp]||c|| ZJJc +|c, C+|p|||||+, C+|||c|||+, uSA. A|| ||c|| ||1J. |c p+|| c| ||| |cc| |+] | |p|cJuJ, cp|J c| ||+|||||J W|||cu| W||||| p||||c|. US $25iiGuido to Flow CytomotryiiiPrinoiplos o Flow CytomotryTabIe of ContentsForeword: An Introduction to FIow Cytometry vEvoIution of FIow Cytometryl| |||ccp |, ] C|||||] |, E|||c|| l|Ju||] Z, Cc|pu|| Z, |cJ|| ||cW C]|c|||] 8CeII BioIogyl| |u|u 6, l| C]|cp|+| 6, l| ||||+| 7, C|| P|p+|+||c| 7FIow Cytometry Instrumentation||cW C]|c||| p|+||c| 9, ||u|J| ||, p|| ||, E|||c|| |8, Sc||W+| |8, l||, R+||c +|J Cc|pu|J P+|+||| |4, l|||p||+||c| c| Auu||J +|+ |4, Sc||||c |4, Au|c|+||c| |6, l|||+| Cc||J|+||c| |6FIuorochromesP|]| c| ||c|| |7, E/||+||c| +|J E|||c| |B, ||uc|c||c| S|||c| |9Antibodiesl||u|cc|c|u||| Z8, Pc|]|c|+| A||||cJ| Z6, |c|c|c|+| A||||cJ| Z7, A||||cJ] A|l|||] ZB, A||||cJ] C|cR+||1||] Z9, A||||cJ] S+|u|+||c| R+| Z9, A||||cJ] S|+|||||] 80, A||||cJ] |+|J|||c 8|Data AnaIysisl|||cJu||c| 88, A|+|]| 88, Cc|p|+||c| 87, +|+ S|c|+c 8B, Cc||u|c| 8BStandardization, CaIibration and QuaIity ControIS|+|J+|J|+||c| 4|, C+||||+||c| 48, u+|||] Cc|||c| 44FIow Sortingl|||cJu||c| 47, P||||p| 47, Y||J, Rc1|] +|J Pu|||] 60, ||c|SpJ Sc||||c 64, V|+|||||] +|J |u|||c| 64Rare-Event Detectionl|||cJu||c| 66, l||||c| 66, Ccu||||c S|+|||| 66, Cc|||||c |c| B+|c|cu|J 67, App|]||c Cc|lJ| ||||| 6B, Sc||||c R+| E1|| 6BImmunophenotypingl|||cJu||c| 69, App||+||c| 60, C||u|+| l||c||+||c| 66, l|||uu 67ivGuido to Flow CytomotryvPrinoiplos o Flow CytomotryTabIe of Contents...oontinuodSmaII-ParticIe AnaIysisl|||cJu||c| 69, App||+||c| 69, l|c|] 70, l|||u||| p||||+||c| 78MHC MuItimers |+|c| |||cc|p+|||||||] Cc|p|/ 76, ||C |u||||| 77, l|||+| Cc||J|+||c| 7BFIuorescent Proteinsl|||cJu||c| B|, u || C]|c|||] BZ, ||uc|| Rc|+| E||c] l|+||| B4, E/p|||||+| P|c|cc| |c| |REl ||cW C]|c|||] B6SignaI Transductionl||||c| +|J 1|1|W B9, |+u|||| c| S|c|+| l|+|Ju||c| E1|| 9|Kineticsl||||c| +|J 1|1|W 98, |+u|||| c| ||||| E1|| 94, |+u|||| c| C+Z-| Rpc| ||||| 96Basic DNA Measurement by FIow Cytometry|A P|c| 99, l||||c|cc] |00, P|c|J] |0|, S+|p| P|p+|+||c| |0|, |A S|+|||||] |0Z, l|||u||| S|up |04, S|+|J+|J|+||c| |04, +|+ A|+|]| +|J Rpc||||c |04, |A +|J B]c|J |06AbsoIute CeII CountingApp||+||c| |07, |||cJ |0B, l|||+| Cc||J|+||c| |||GIossary ||6Index ||7Foreword: An Introduction to FIow CytometrySonja Wulff, EditorFlow oytomotry is a oomplox hold tnat draws pooplo rom divorso soiontiho baokgrounds. Wnotnor you'ro an immunologist or aquatio ooologist, rosoaronor or olinioian, wo nopo tnis guidobook will nolp you in your journoy o disoovoring tno poworul toonnology and applioations o how oytomotry.Tnoso o you wno aro alroady amiliar witn how oytomotry may want to skip dirootly to Cnaptor 1. Howovor, or tnoso o you wno would liko a littlo moro baokground booro jumping into tno languago o orward soattor, huoroonromos and FPET, koop roading: Flow oytomotry is tno soionoo o moasuring pnysioal and onomioal proportios o livo oolls or otnor biologioal partiolos as tnoy pass in a huid, singlo-ooll stroam tnrougn a moasuring apparatus. n tno most oommon soonario, ono or moro lasors intorrogato oaon partiolo and, at a minimum, tno systom moasuros tno dogroo and dirootion o soattorod lignt indioators o tno partiolo's sizo, snapo and struoturo. partiolos navo boon stainod witn ono or moro huorosoont dyos known as huoroonromos tno lignt souroo oxoitos tnoso dyos to provido additional biologioal inormation about oaon partiolo, suon as motabolio aotivity, DNA oontont and tno prosonoo o spooiho suraoo and intraoollular markors. Prooiso optioal and olootronio olomonts oolloot tno huorosoont pulsos and soattorod lignt, oonvort tnom into digital valuos and sond tnom to a oomputor or analysis. Somo how oytomotors aro also oquippod to idontiy and sort usor-spooihod partiolos into oollootion vossols. Hign-porormanoo ooll sortors oan routinoly roaon ratos o 70,000 oolls por sooond.Tno uniquo powor o how oytomotors is tnat tnoy oan rapidly and quantitativoly moasuro multiplo simultanoous paramotors on individual livo oolls and tnon isolato oolls o intorost. Additionally, tno sonsitivity and tnrougnput ratos aoniovablo by nign-porormanoo oommoroial instrumonts onablo dotootion o oxtromoly raro populations and ovonts (roquonoios bolow 10-6), suon as stom oolls, dondritio oolls, antigon-spooiho T oolls and gonotio transootants.1 As a rosult, applioations or how oytomotors oontinuo to grow. n addition to traditional immunology and patnology applioations involving partiolos suon as lympnooytos, maoropnagos, monooytos and tumor oolls, how oytomotors aro widoly usod in oonjunotion witn huorosoonoo-basod protoin roportors, suon as groon huorosoont protoin (GFP). n tnis arona, how oytomotors oan monitor botn transootion ohoionoy and protoin oxprossion lovols.2-3 Tnoy also oan dotoot huorosoonoo rosonanoo onorgy transor (FPET), wnion providos inormation about molooular intoraotions, protoin struoturo and DNA soquonoo.4-5 ntorost is growing in tno uso o how oytomotors to soroon ooll- or boad-basod oombinatorial librarios.6 noroasingly, how oytomotrio assays aro usod to dotoot molooulos tnat bind to a targot protoin in vitro or oxnibit a partioular aotivity in a ooll-basod assay. Flow oytomotry also onablos sorooning o protoin librarios oxprossod viGuido to Flow CytomotryviiPrinoiplos o Flow Cytomotryin oolls or displayod on tno suraoo o baotoria or boads. A how oytomotor, or instanoo, oan dotoot modulation o a signal transduotion patnway by a partioular small molooulo7-8 and idontiy protoins witn a partioular binding spooihoity, onzymatio aotivity, oxprossion lovol and stability.9-12 Witn any applioation, oolls oxnibiting dosirod aotivity prohlos oan bo sortod into tost tubos, multiwoll platos or miorosoopo slidos. At purity ratos groator tnan 99%, tnoso sortod partiolos aro tnon availablo or urtnor studios suon as PCP analysis or in situ nybridization,13 and tno oolls aro ully unotional or long-torm ox vivo oulturo, oxpansion, transplantation or otnor subsoquont applioations.14-15 Ongoing dovolopmont oorts in tno how oytomotry industry aro aimod at automation and laboratory intogration. nput/output robotios, pusnbutton oporation and automatod samplo proparation will inoroaso tnrougnput ratos and mako tno toonnology moro aooossiblo to a widor usor baso, as now huorosoont dyos and oroativo sorooning approaonos oxpand applioations into tno protoomio arona. Evontually, sotwaro advanoos will soamlossly notwork instrumonts into oomprononsivo analytioal and diagnostio systoms, and tno industry may marry its toonnology witn imaging and miorohuidios. Obviously, tnis is only a vory suporhoial disoussion o tno hold o how oytomotry. t is intondod moroly to sot tno stago or tno rost o tno publioation, wnion will introduoo you to moro dotails about tno toonnology and applioations o how oytomotry. Wo, at Dako, aro nonorod to bo part o your oxploration. EnjoyReferences1. Asnorot PG, Lopoz PA. Commoroial nign spood maoninos opon now opportunitios in nign tnrougnput how oytomotry. J mmunol Motnods 2000; 243:13.2. Mao X, Fujiwara Y, Cnapdolaino A, Yang H, Orkin SH. Aotivation o EGFP oxprossion by Cro-modiatod oxoision in a now POSA26 roportor mouso strain. Blood 2001; 97: 324.3. Poson ED, Husi C, Wang X, Sakai S, Frooman MW, Gonzaloz FJ ot al. C/EBP! induoos adipogonosis tnrougn PPAP: a unihod patnway. Gonos & Dovolop 2002; 16, 22.4. Ponoarati P, Sostan N, Sonoinold MH, Boroonid BE, Lopoz PA, Mouooi O. ot al. 2002. Tno !-soorotaso-gonoratod intraoollular domain o "-amyloid prooursor protoin binds Numb and innibits Noton signaling. PNAS 2002; 99: 7102.5. Sonoinold MH, Ponoarati P, vito P, Lopoz PA, Abdallan M, D'Adamio L. Jun NH2-torminal kinaso (JNK) intoraoting protoin 1 (JP1) binds tno oytoplasmio domain o tno Alznoimor's "-amyloid prooursor protoin (APP). J Biol Cnom 2002; 277: 3767.6. Daugnorty PS, vorson BL, Goorgiou G. Flow oytomotrio sorooning o ooll-basod librarios. J mmunol Motnods 2000; 243: 211.7. Domo SD, Masuda E, Possi AB, Tnrondsot BT, Gorard A, Cnan EH ot al. Quantitativo moasuromont o mast ooll dogranulation using a novol how oytomotrio annoxin-v binding assay. Cytomotry 1999; 36: 340.8. Lorons JB, Bonnott MK, Poarsall DM, Tnrondsot WP, Possi AB, Armstrong PJ ot al. Potroviral dolivory o poptido modulators o oollular unotions. Molooular Tnorapy 2000; 1: 438.9. Wontzol A, Cnristmann A, Adams T, Kolmar H. Display o passongor protoins on tno suraoo o &TDIFSJDIJBDPMJK-12 by tno ontoronomorrnagio &DPMJntimin EaoA. J Baotoriol 2001; 183: 7273.10. Wontzol A, Cnristmann A, Kratznor P, Kolmar H. Soquonoo roquiromonts o tno GPNG "-turn o tno &DCBMMJVNFMBUFSJVNtrypsin innibitor oxplorod by oombinatorial library soro