Ficoll-PaquePLUSInstructions71-7167-00AGCellpreparationGEHealthcareFicoll-PaqueIntendedUseForinvitroisolationoflymphocytes.IntroductionandSummaryoftheMethodThereisaneedforarapid,simpleandreliablemethodofisolatinglymphocytesfromwholebloodinsuchareasofscientificendeavorashistocompatibilitytesting,invitrocellmediatedimmunityassays,andotherproceduresrequiringpurelymphocytepreparations.Earlytechniquesforseparatinglymphocytesfromotherbloodcelltypesinvolvedmixingthebloodwithsomeerythrocyteaggregatingagent,therebycausingtheerythrocytestoclumpandsedimenttothebottomofthetube.Thelymphocytescouldthenbecollectedfromtheupperpartofthetube(1,2).Thedisadvantagesofthesetechniquesarethatlengthy,repeatedproceduresarerequiredtoobtainpurifiedlymphocytesuspensions,andtheyieldofrecoveredlymphocytesislow.BøyumnotedthatthelowviscosityofFicollTM,comparedtotheotherpolymericerythrocyteaggregatingagents,makesitpossibletodevisealymphocyteisolationprocedureinvolvingashort,lowspeedcentrifugation(3).AsolutionofFicollandsodiummetrizoateoftheproperdensityandosmoticstrengthwasplacedinacentrifugetube.Bloodwaslayeredontop,andthetwo-phasesystemwascentrifugedatalowspeedforashorttime.Theerythrocytesandgranulocytessedimentedtothebottomofthetube,andthepurifiedlymphocytescouldbecollectedfromtheinterfacebetweenthetwophases.Otherauthors,usingslightmodificationsofthisprocedure,havep.2Contents1.PrincipleoftheProcedure32.Reagents43.Precautions44.Storage45.IndicationsofInstability46.SpecimenCollectionandHandling47.Procedure58.ParametersoftheMethod59.PreparationofReagents610.TestProtocol711.ExpectedValue1012.FactorsAffectingLymphocyteIsolationbytheFicoll-PaquePLUSProcedure1013.HowSupplied1114.OrderingInformation1115.References12p.3emphasizedthattheFicoll-sodiummetrizoatecentrifugationprocedureisaneasyone-step,rapid,reproduciblemethodforthepreparationofviablelymphocytesinhighyield(4–7).Sodiumdiatrizoatehasbeensuccessfullysubstitutedforsodiummetrizoateinthisprocedurebynumerousworkers(8–11).Ficoll-PaqueTMPLUSprovi...
