Percoll密度梯度離心教程第1頁，共20頁。OutlinePrinciplesPhysicalpropertiesStorageMakeandusedensityofPercollTipsProtocolInstrumentandoperationReferencesandapplicationsContrast(differentialvelocitycentrifugation)第2頁，共20頁。Forbiologicalparticles,theidealgradientmediumhasbeendescribedasonehavingthefollowingcharacteristics:?coversasufficientdensityrangeforisopycnicbandingofallbiologicalparticlesofinterest?possessesphysiologicalionicstrengthandpH?isiso-osmoticthroughoutthegradient?haslowviscosity?isnon-toxic?willnotpenetratebiologicalmembranes?issuppliedsterileandisresterilizable?willformself-generatedgradientsbycentrifugationatmoderateg-forces?iscompatiblewithbiologicalmaterials?iseasilyremovedfrompurifiedmaterials?doesnotaffectassayprocedures?willnotquenchradioactiveassays第3頁，共20頁。Principlesofdensitygradientcentrifugation

第4頁，共20頁。Separationbydensity(isopycniccentrifugation)Separationbysize(ratezonalcentrifugation)第5頁，共20頁。Percoll–physicalpropertiesPercollisavailablefromGEHealthcare.Compositionsilicasolwithnondialyzablepolyvinylpyrrolidone(PVP)coating（由聚乙烯吡咯烷酮包裹的硅膠顆粒）Density1.130±0.005g/mlConductivity1.0mS/cm（電導率）Osmolality<25mOsm/kgH2O（滲透壓）Viscosity10±5cPat20°C（粘度）pH9.0±0.5at20°CRefractiveIndex1.3540±0.005at20°C（折射率）Percollisnon-toxic1.0~1.3g/mlTheviscosityofPercollislowerinsalinesolutionsatphysiologicalionic第6頁，共20頁。StorageofPercollSterileandunopen----5yearsWhenopened,Percollshouldbestoredbelow+8℃IfPercollopenedundernon-sterileconditions,itcanbefrozenforupto6minthsat-18℃Preformedgradientscanbestoredforweekswithoutachangeingradientshape,providedthatthegradientissterileandisnotphysicallydisturbedIfstoredat-18°C,gradientsformuponthawing,necessitatingamixingofthecontentsofthebottlebeforeuse.第7頁，共20頁。SterilizationofPercollsolutions120°Cfor30minAbsenceofsaltsorsucrose.Minimumcontactwithair(narrow-neckedbottle)IfPercollformparticles,theseparticlesmayberemovedbylowspeedcentrifugation.Ifanysignificantevaporationoccursduringautoclaving,thevolumeshouldbereplenishedwithsterilewatersothatthedensityisnotaffected.第8頁，共20頁。HowtomakeandusegradientsofPercoll

—MakinganddilutingastocksolutionofPercollWhereVx=volumeofdilutingmedium(ml)Vo=volumeofundilutedPercoll(ml)ρo=densityofPercoll(1.130+0.005g/ml*)ρ10=densityof1.5MNaCl=1.058g/mldensityof2.5Msucrose=1.316g/mlρi=densityofSIPsolutionproduced(g/ml)Thus,forSIPinsaline,ρi=1.123g/ml,forSIPinsucrose,ρi=1.149g/ml,assumingρo=1.130g/ml.Adding9parts(v/v)ofPercollto1part(v/v)of1.5MNaClor10×concentratedcellculturemediumisasimplewayofpreparingaStockIsotonicPercoll(SIP)solution.“滲透活性物質的物質的量除以溶液的體積稱為溶液的滲透濃度（osmolarity），單位為mol/L或mmol/L”第9頁，共20頁。HowtomakeandusegradientsofPercoll

—DilutingstocksolutionstolowerdensitiesSolutionsofStockIsotonicPercoll(SIP)aredilutedtolowerdensitiessimplybyadding0.15MNaCl(ornormalstrengthcellculturemedium)forcellwork,orwith0.25Msucrosewhenworkingwithsubcellularparticlesorviruses.第10頁，共20頁。TipsofmakingandusegradientsofPercollThecolloiddoesnotperceptiblydiffuseovertime,resultingintheformationofverystablegradients.Therefore,bothdiscontinuousandcontinuousgradientscanbepreparedweeksinadvance,givinggreatreproducibilityoverthecourseofanexperiment.Measuretheweightofthesolution,makesuretheweightofsolutionareallthesame.實驗重復性的保證WeightofPercoll50%55%60%65%SIP-20170914SIP-20171014SIP-20171114第11頁，共20頁。1.Percollwasdiluted9:1(vol/vol)with1.5MNaCl,2.Put10mlofthePercollsolutioninto15-mlCorextubesandcentrifugedat19,240gavfor15minat20°C.(swingingbucketrotor)3.Approximately2×109cells(200OD600)werepelleted,resuspendedin1mlTrisbuffer,overlaidontothepreformedgradient,andcentrifugedat400gavfor60min.Isolationofquiescentandnonquiescentcellsformyeaststationary-phasecultureAllenC.TheJournalofcellbiology

.2006第12頁，共20頁。PercollgradientpurificationofsporesStrainsweregrownovernightinYPDliquidmedium.CellswerewashedtwicewithddH2OanddilutedtoafinalcelldensityofOD600=0.5.Then,10μlofequal-volumemixedcellswerespottedonV8mediumandincubatedforsevendaysinthedarkatroomtemperature.Theentirematingpatchwassuspendedin60%Percoll(GEHealth)inPBSwith0.1%TritonX100.Aftercentrifugationat10,000Xgfor30minsinanSW41Tiultracentrifugerotor(Beckman-Coulter),abandofsporesnearthebottomofthePercollgradientwasrecoveredwitha1-mltuberculinsyringeandtransferredintoanEppendorftube.Thetotalsporeproductionwasdeterminedbymultiplyingthesporedensity,measuredbyhemocytometer,withthefinalvolume.ChristinaM.HullPLoSgenetics.2015第13頁，共20頁。Instrumentandoperation第14頁，共20頁。Instrumentandoperation第15頁，共20頁。Instrumentandoperation第16頁，共20頁。Instrumentandoperation

—dataexport第17頁，共20頁。Referencesan