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Cell Biology Protocols Cell B iology P rotocols . E dited by J . Robin H arris, John Graham, D avid Rickwood  2006 John Wiley & Sons, L td. ISBN: 0-470-84758-1 Cell Biology Protocols Editors J. Robin Harris Institute of Zoology, Johannes Gutenberg-Universita¨t, Mainz, Germany John Graham JG Research Consultancy, Upton, Wirral, UK David Rickwood Department of Biological Sciences, University of Essex, Colchester, UK Copyright2006 JohnWiley&SonsLtd,TheAtrium,SouthernGate,Chichester, WestSussexPO198SQ,England Telephone(+44)1243779777 Email(forordersandcustomerserviceenquiries):[email protected] VisitourHomePageonwww.wiley.com AllRightsReserved.Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedin anyformorbyanymeans,electronic,mechanical,photocopying, recording,scanningorotherwise,exceptunder thetermsoftheCopyright,DesignsandPatentsAct1988orunderthetermsofalicenceissuedbytheCopyright LicensingAgencyLtd,90TottenhamCourtRoad,LondonW1T4LP,UK,withoutthepermissioninwritingof thePublisher.RequeststothePublishershouldbeaddressedtothePermissionsDepartment,JohnWiley&Sons Ltd,TheAtrium,SouthernGate,Chichester,WestSussexPO198SQ,England,oremailedto [email protected],orfaxedto(+44)1243770620. 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OtherWileyEditorialOffices JohnWiley&SonsInc.,111RiverStreet,Hoboken,NJ07030,USA Jossey-Bass,989MarketStreet,SanFrancisco,CA94103-1741,USA Wiley-VCHVerlagGmbH,Boschstr.12,D-69469Weinheim,Germany JohnWiley&SonsAustraliaLtd,42McDougallStreet,Milton,Queensland4064,Australia JohnWiley&Sons(Asia)PteLtd,2ClementiLoop#02-01,JinXingDistripark,Singapore129809 JohnWiley&SonsCanadaLtd,22WorcesterRoad,Etobicoke,Ontario,CanadaM9W1L1 Wileyalsopublishesitsbooksinavarietyofelectronicformats.Somecontentthatappears inprintmaynotbeavailableinelectronicbooks. LibraryofCongressCataloging-in-PublicationData: Cellbiologyprotocols/editors,J.RobinHarris,JohnGraham,DavidRickwood. p.cm. ISBN-13:978-0-470-84758-1 ISBN-10:0-470-84758-1 1. Cytology–Laboratorymanuals. I.Harris,JamesR. II.Graham,J.M.(JohnM.), 1943-III.Rickwood,D.(David) QH583.2.C472005 571.6078–dc22 2005029713 BritishLibraryCataloguinginPublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary ISBN-13:978-0-470-84758-1 ISBN-10:0-470-84758-1 Typesetin10.5/12.5ptTimesbyLaserwordsPrivateLimited,Chennai,India PrintedandboundinGreatBritainbyAntonyRoweLtd,Chippenham,Wiltshire Thisbookisprintedonacid-freepaperresponsiblymanufacturedfromsustainableforestry inwhichatleasttwotreesareplantedforeachoneusedforpaperproduction. Contents Preface xi List of Contributors xiii 1 Basic Light Microscopy 1 Minnie O’Farrell Introduction 1 Keycomponentsofthecompoundmicroscope 2 Techniquesofmicroscopy 6 Protocols 1.1 Settingupthemicroscopeforbrightfieldmicroscopy 7 1.2 SettingKo¨hlerillumination 8 1.3 Focusingprocedure 9 1.4 Settingupthemicroscopeforphasecontrastmicroscopy 11 1.5 Settingupthemicroscopeforepifluorescence 14 1.6 Poly-L-lysinecoating 18 References 19 2 Basic Electron Microscopy 21 J. Robin Harris Introduction 21 EMmethodsavailable 22 Protocols 2.1 Preparationofcarbon-formvar,continuouscarbonandholeycarbonsupportfilms 25 2.2 The‘droplet’negativestainingprocedure(usingcontinuouscarbon,formvar–carbon andholeycarbonsupportfilms) 27 2.3 Immunonegativestaining 29 2.4 Thenegativestaining-carbonfilmtechnique:cellandorganellecleavage 31 2.5 Preparationofunstainedandnegativelystainedvitrifiedspecimens 33 2.6 Metalshadowingofbiologicalspecimens 35 2.7 Aroutineschedulefortissueprocessingandresinembedding 37 2.8 Agaroseencapsulationforcellandorganellesuspensions 39 2.9 Routinestainingofthinsectionsforelectronmicroscopy 40 2.10 Post-embeddingindirectimmunolabellingofthinsections 42 2.11 Imagingthenuclearmatrixandcytoskeletonbyembedment-freeelectronmicroscopy 44 JeffreyA.NickersonandJeanUnderwood References 50 vi CONTENTS 3 Cell Culture 51 Anne Wilson and John Graham Cells:isolationandanalysis 51 AnneWilson Mechanicaldisaggregationoftissue 52 Protocols 3.1 Tissuedisaggregationbymechanicalmincingorchopping 54 3.2 Tissuedisaggregationbywarmtrypsinization 56 3.3 Coldtrypsinization 58 3.4 Disaggregationusingcollagenaseordispase 60 AnneWilson 3.5 Recoveryofcellsfromeffusions 63 AnneWilson 3.6 Removalofredbloodcellsbysnaplysis 64 3.7 Removalofredbloodcellsanddeadcellsusingisopycniccentrifugation 65 AnneWilson 3.8 Quantitationofcellcountsandviability 67 AnneWilson 3.9 Recoveryofcellsfrommonolayercultures 71 AnneWilson 3.10 Freezingcells 74 3.11 Thawingcells 76 JohnGraham 3.12 PurificationofhumanPBMCsonadensitybarrier 80 3.13 PurificationofhumanPBMCsusingamixertechnique 82 3.14 PurificationofhumanPBMCsusingabarrierflotationtechnique 83 References 84 4 Isolation and Functional Analysis of Organelles 87 John Graham Introduction 88 Homogenization 88 Differentialcentrifugation 90 Densitygradientcentrifugation 91 Nucleiandnuclearcomponents 92 Mitochondria 93 Lysosomes 94 Peroxisomes 94 Roughandsmoothendoplasmicreticulum(ER) 95 Golgimembranes 96 Plasmamembrane 96 Chloroplasts 97 Protocols 4.1 Isolationofnucleifrommammalianliverinaniodixanolgradient(withnoteson culturedcells) 98 4.2 Isolationofmetaphasechromosomes 100 CONTENTS vii 4.3 Isolationofthenuclearenvelope 102 4.4 Nuclearporecomplexisolation 104 J.RobinHarris 4.5 Preparationofnuclearmatrix 106 4.6 Preparationofnucleoli 107 4.7 Isolationofaheavymitochondrialfractionfromratliverbydifferential centrifugation 108 4.8 Preparationofalightmitochondrialfractionfromtissuesandculturedcells 110  4.9 PurificationofyeastmitochondriainadiscontinuousNycodenz gradient 112 4.10 Purificationofmitochondriafrommammalianliverorculturedcellsina  median-loadeddiscontinuousNycodenz gradient 114 4.11 Succinate–INTreductaseassay 116  4.12 IsolationoflysosomesinadiscontinuousNycodenz gradient 117 4.13 β-Galactosidase(spectrophotometricassay) 119 4.14 β-Galactosidase(fluorometricassay) 120 4.15 Isolationofmammalianperoxisomesinaniodixanolgradient 121 4.16 Catalaseassay 123 4.17 Analysisofmajororganellesinapreformediodixanolgradient 124 4.18 SeparationofsmoothandroughERinpreformedsucrosegradients 127 4.19 SeparationofsmoothandroughERinaself-generatediodixanolgradient 129 4.20 NADPH-cytochromecreductaseassay 131 4.21 Glucose-6-phosphataseassay 132 4.22 RNAanalysis 133 4.23 IsolationofGolgimembranesfromliver 134 4.24 AssayofUDP-galactosegalactosyltransferase 136 4.25 Purificationofhumanerythrocyte‘ghosts’ 137 4.26 Isolationofplasmamembranesheetsfromratliver 139 4.27 Assayfor5(cid:1)-nucleotidase 141 4.28 Assayforalkalinephosphodiesterase 143 4.29 Assayforouabain-sensitiveNa+/K+-ATPase 144 4.30 Isolationofchloroplastsfromgreenleavesorpeaseedlings 145 4.31 Measurementofchloroplastchlorophyll 147 4.32 Assessmentofchloroplastintegrity 148 5 Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling 153 John Graham Introduction 154 Methodsavailable 154 Plasmamembranedomains 155 Analysisofmembranecompartmentsintheendoplasmicreticulum–Golgi–plasma membranepathway 156 Separationofmembranevesiclesfromcytosolicproteins 157 Endocytosis 158 Protocols 5.1 Separationofbasolateralandbilecanalicularplasmamembranedomainsfrom mammalianliverinsucrosegradients 160 viii CONTENTS 5.2 Isolationofratliversinusoidaldomainusingantibody-boundbeads 162 5.3 FractionationofapicalandbasolateraldomainsfromCaco-2cellsinasucrose gradient 163 5.4 FractionationofapicalandbasolateraldomainsfromMDCKcellsinaniodixanol gradient 165 5.5 Isolationoflipidrafts 167 5.6 Isolationofcaveolae 170 5.7 AnalysisofGolgiandERsubfractionsfromculturedcellsusingdiscontinuous sucrose–D Odensitygradients 172 2 5.8 AnalysisofGolgi,ER,ERGICandothermembranecompartmentsfromculturedcells usingcontinuousiodixanoldensitygradients 174 5.9 AnalysisofGolgi,ER,TGNandothermembranecompartmentsinsedimentation velocityiodixanoldensitygradients(continuousordiscontinuous) 177 5.10 SDS–PAGEofmembraneproteins 180 5.11 Semi-dryblotting 182 5.12 Detectionofblottedproteinsbyenhancedchemiluminescence(ECL) 183 5.13 Separationofmembranesandcytosolicfractionsfrom(a)mammaliancellsand(b) bacteria 185 5.14 Analysisofearlyandrecyclingendosomesinpreformediodixanolgradients; endocytosisoftransferrinintransfectedMDCKcells 188 5.15 Analysisofclathrin-coatedvesicleprocessinginself-generatediodixanolgradients; endocytosisofasialoglycoproteinbyratliver 191  5.16 Polysucrose–Nycodenz gradientsfortheanalysisofdenseendosome–lysosome eventsinmammalianliver 194 References 196 6 In Vitro Techniques 201 Edited by J. Robin Harris Introduction 203 Protocols Nuclearcomponents 6.1 NucleosomeassemblycoupledtoDNArepairsynthesisusingahumancellfreesystem 204 Genevie`veAlmouzniandDorisKirschner 6.2 SinglelabellingofnascentDNAwithhalogenatedthymidineanalogues 210 DanielaDimitrova 6.3 DoublelabellingofDNAwithdifferenthalogenatedthymidineanalogues 214 6.4 Simultaneousimmunostainingofproteinsandhalogen-dU-substitutedDNA 217 6.5 Uncoveringthenuclearmatrixinculturedcells 220 JeffreyA.Nickerson,JeanUnderwoodandStefanWagner 6.6 Nuclearmatrix–lamininteractions:invitroblotoverlayassay 228 BarbaraKorbeiandRolandFoisner 6.7 Nuclearmatrix–lamininteractions:invitronuclearreassemblyassay 230 6.8 PreparationofXenopuslaeviseggextractsandimmunodepletion 234 TobiasC.Walther 6.9 Nuclearassemblyinvitroandimmunofluorescence 237 MartinHetzer 6.10 NucleocytoplasmictransportmeasurementsusingisolatedXenopusoocytenuclei 240 ReinerPeters 6.11 Transportmeasurementsinmicroarraysofnuclearenvelopepatchesbyopticalsingle transporterrecording 244 ReinerPeters CONTENTS ix Cellsandmembranesystems 6.12 CellpermeabilizationwithStreptolysinO 248 IvanWalev 6.13 Nanocapsules:anewvehicleforintracellulardeliveryofdrugs 250 AntonI.P.M.deKroon,RutgerW.H.M.Staffhorst,BendeKruijffandKoertN.J. Burger 6.14 Arapidscreenfordeterminationoftheprotectiveroleofantioxidantproteinsin yeast 255 LuisEduardoSoaresNetto 6.15 Invitroassessmentofneuronalapoptosis 259 EricBertrand 6.16 Themitochondrialpermeabilitytransition:PTand(cid:1)(cid:2)mlossdeterminedincellsor isolatedmitochondriawithconfocallaserimaging 265 JudieB.AlimontiandArnoldH.Greenberg 6.17 Themitochondrialpermeabilitytransition:measuringPTand(cid:1)(cid:2)mlossinisolated mitochondriawithRh123inafluorometer 268 JudieB.AlimontiandArnoldH.Greenberg 6.18 Themitochondrialpermeabilitytransition:measuringPTand(cid:1)(cid:2)mlossincellsand isolatedmitochondriaontheFACS 270 JudieB.AlimontiandArnoldH.Greenberg 6.19 MeasuringcytochromecreleaseinisolatedmitochondriabyWesternblotanalysis 271 JudieB.AlimontiandArnoldH.Greenberg 6.20 Proteinimportintoisolatedmitochondria 272 JudieB.AlimontiandArnoldH.Greenberg 6.21 FormationofternarySNAREcomplexesinvitro 274 JinnanXiao,AnuradhaPradhanandYuechuengLiu 6.22 Invitroreconstitutionofliverendoplasmicreticulum 277 JacquesPaiementandRobinYoung 6.23 Asymmetricincorporationofglycolipidsintomembranesanddetectionoflipid flip-flopmovement 280 Fe´lixM.Gon˜i,Ana-VictoriaVillar,F.-XabierContrerasandAliciaAlonso 6.24 Purificationofclathrin-coatedvesiclesfromratbrains 286 BrianJ.PeterandIanG.Mills 6.25 Reconstitutionofendocyticintermediatesonalipidmonolayer 288 BrianJ.PeterandMatthewK.Higgins 6.26 Golgimembranetubuleformation 293 WilliamJ.Brown,K.ChambersandA.Doody 6.27 Tightjunctionassembly 296 C.YanChengandDoloresD.Mruk 6.28 Reconstitutionofthemajorlight-harvestingchlorophylla/bcomplexintoliposomes 300 ChunhongYang,HelmutKirchhoff,WinfriedHaase,StephanieBoggaschandHarald Paulsen 6.29 Reconstitutionofphotosystem2intoliposomes 305 JulieBenesova,Sven-T.LiffersandMatthiasRo¨gner 6.30 Golgi–vimentininteractioninvitroandinvivo 307 Ya-shengGaoandElizabethSztul Cytoskeletalandfibrillarsystems 6.31 Microtubuleperoxisomeinteraction 313 MeinolfThiemannandH.DariushFahimi 6.32 Detectionofcytomatrixproteinsbyimmunogoldembedment-freeelectron microscopy 317 RobertGniadeckiandBarbaraGajkowska x CONTENTS 6.33 Tubulinassemblyinducedbytaxolandothermicrotubuleassemblypromoters 326 SusanL.Bane 6.34 Vimentinproduction,purification,assemblyandstudybyEPR 331 JohnF.Hess,JohnC.VossandPaulG.FitzGerald 6.35 Neurofilamentassembly 337 Shin-ichiHisanagaandTakahiroSasaki 6.36 α-Synucleinfibrilformationinducedbytubulin 342 KenjiUe´daandShin-ichiHisanaga 6.37 Amyloid-βfibrilformationinvitro 345 J.RobinHarris 6.38 SolubleAβ peptideinducestauhyperphosphorylationinvitro 348 1–42 TerrenceTownandJunTan 6.39 Anti-sensepeptides 353 NathanielG.N.Milton 6.40 Interactionsbetweenamyloid-βandenzymes 359 NathanielG.N.Milton 6.41 Amyloid-βphosphorylation 364 NathanielG.N.Milton 6.42 Smitin–myosinIIcoassemblyarraysinvitro 369 RichardChiandThomasC.S.KellerIII 6.43 Assembly/disassemblyofmyosinfilamentsinthepresenceofEF-hand calcium-bindingproteinS100A4invitro 372 MarinaKriajevska,IgorBronsteinandEugeneLukanidin 6.44 Collagenfibrilassemblyinvitro 375 DavidF.HolmesandKarlE.Kadler 7 Selected Reference Data for Cell and Molecular Biology 379 David Rickwood Chemicalsafetyinformation 379 Centrifugationdata 386 Radioisotopedata 388 Index 391