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PercolldensitygradientcentrifugationOutline•Principles•Physicalproperties•Storage•MakeandusedensityofPercoll•Tips•Protocol•Instrumentandoperation•Referencesandapplications•Contrast(differentialvelocitycentrifugation)•Forbiologicalparticles,theidealgradientmediumhasbeendescribedasonehavingthefollowingcharacteristics:•coversasufficientdensityrangeforisopycnicbandingofallbiologicalparticlesofinterest•possessesphysiologicalionicstrengthandpH•isiso-osmoticthroughoutthegradient•haslowviscosity•isnon-toxic•willnotpenetratebiologicalmembranes•issuppliedsterileandisresterilizable•willformself-generatedgradientsbycentrifugationatmoderateg-forces•iscompatiblewithbiologicalmaterials•iseasilyremovedfrompurifiedmaterials•doesnotaffectassayprocedures•willnotquenchradioactiveassaysPrinciplesofdensitygradientcentrifugationTheequationforthesedimentationofasphereinacentrifugalfieldis:V=𝑑2(ρp−ρl)18η×𝑔wherev=sedimentationrated=diameteroftheparticle(hydrodynamicallyequivalentsphere)ρp=particledensityρl=liquiddensityη=viscosityofthemediumg=centrifugalforceSeparationbydensity(isopycniccentrifugation)Separationbysize(ratezonalcentrifugation)Percoll–physicalproperties•PercollisavailablefromGEHealthcare.•Compositionsilicasolwithnondialyzablepolyvinylpyrrolidone(PVP)coating（由聚乙烯吡咯烷酮包裹的硅胶颗粒）•Density1.130±0.005g/ml•Conductivity1.0mS/cm（电导率）•Osmolality25mOsm/kgH2O（渗透压）•Viscosity10±5cPat20°C（粘度）•pH9.0±0.5at20°C•RefractiveIndex1.3540±0.005at20°C（折射率）•Percollisnon-toxic1.0~1.3g/mlTheviscosityofPercollislowerinsalinesolutionsatphysiologicalionicStorageofPercoll•Sterileandunopen----5years•Whenopened,Percollshouldbestoredbelow+8℃•IfPercollopenedundernon-sterileconditions,itcanbefrozenforupto6minthsat-18℃•Preformedgradientscanbestoredforweekswithoutachangeingradientshape,providedthatthegradientissterileandisnotphysicallydisturbed•Ifstoredat-18°C,gradientsformuponthawing,necessitatingamixingofthecontentsofthebottlebeforeuse.SterilizationofPercollsolutions•120°Cfor30min•Absenceofsaltsorsucrose.•Minimumcontactwithair(narrow-neckedbottle)•IfPercollformparticles,theseparticlesmayberemovedbylowspeedcentrifugation.•Ifanysignificantevaporationoccursduringautoclaving,thevolumeshouldbereplenishedwithsterilewatersothatthedensityisnotaffected.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.“渗透活性物质的物质的量除以溶液的体积称为溶液的渗透浓度（osmolarity），单位为mol/L或mmol/L”HowtomakeandusegradientsofPercoll—Dilutingstocksolutionstolowerdensities•SolutionsofStockIsotonicPercoll(SIP)aredilutedtolowerdensitiessimplybyadding0.15MNaCl(ornormalstrengthcellculturemedium)forcellwork,orwith0.25Msucrosewhenworkingwithsubcellularparticlesorviruses.TipsofmakingandusegradientsofPercoll•Thecolloiddoesnotperceptiblydiffuseovertime,resultingintheformationofverystablegradients.Therefore,bothdiscontinuousandcontinuousgradientscanbepreparedweeksinadvance,givinggreatreproducibilityoverthecourseofanexperiment.•Measuretheweightofthesolution,makesuretheweightofsolutionareallthesame.实验重复性的保证WeightofPercoll50%55%60%65%SIP-20170914SIP-20171014SIP-201711141.Percollwasdiluted9:1(vol/vol)with1.5MNaCl,2.Put10mlofthePercollsolutioninto15-mlCorextubesandcentrifugedat19,240gavfor15minat20°C.(swingingbucketrotor)3.Approximately2×109cells(200OD600)werepelleted,resuspendedin1mlTrisbuffer,overlaidontothepreformedgradient,andcentrifugedat400gavfor60min.Isolationofquiescentandnonquiescentcellsformyeaststationary-phasecultureAllenC.TheJournalofcellbiology.2006Percollgradientpurificationofspores•StrainsweregrownovernightinYPDliquidmedium.CellswerewashedtwicewithddH2OanddilutedtoafinalcelldensityofOD600=0.5.•Then,10μlofequal-volumemixedcellswerespottedonV8mediumandincubatedforsevendaysinthedarkatroomtemperature.•Theentirematingpatchwassuspendedin60%Percoll(GEHealth)inPBSwith0.1%TritonX100.•Aftercentrifugationat10,000Xgfor30minsinanSW41Tiultracentrifugerotor(Beckman-Coulter),abandofsporesnearthebottomofthePercollgradientwasrecoveredwitha1-mltuberculinsyringeandtransferredintoanEppendorftube.•Thetotalsporeproductionwasdeterminedbymultiplyingthesporedensity,measuredbyhemocytometer,withthefinalvolume.ChristinaM.HullPLoSgenetics.2015InstrumentandoperationInstrumentandoperationInstrumentandoperationInstrumentandoperation—dataexportReferencesandapplicationscontrastdifferentialvelocitycentrifugationdensitygradientcentrifugationSeparationcriteriaWeightDensityCentrifugalspeedTwoormoreOnlyoneSuitablematerialsDifferencesindensityDifferencesinsedimentationcoefficient