CodeNo.143-07041(1g10)WakoNylonFiberColumnT(L-Type)IntroductionFortheresearchinimmunesystemsinvivoandinvitro,acrucialprocedureisseparationoflymphocytescellpopulations,consistingofavarietyofcellssuchasTcellsandBcells.NylonFiberColumnT(L-Type)isoptimizedonthebasisofaTcellpurificationmethod,developedbyMasuda,Julius1)etal.ThisutilizestheprincipleofNylonFibersaffinitytoBcells,thusprovidingTcellpreparationsofsufficientpurityandwithoutseveredamages,throughpreferentialabsorptionofBcellstothefiberinthecolumn.TheNylonFiberColumnT(L-Type)issuitableforalargeamountofsuspendedsolution,especiallyratspleniclymphocytes,andperipheralbloodlymphocytesofrabbitandhuman.YouareabletouseNylonFiberColumnT(147-06721)whenyousepareteTcellsofmouselymphocytes.Procedure1.Sample4108cells/mLpreparedfromratspleens;usuallycontainsTcellsat30-35%andBcellsat55-65%.Cellsuspensioncontaining22.ProcedureStep1:WashingNylonFiberColumnT(L-Type)Washingthecolumnwith20mLMEM,warmedat37inordertoequilibrateNylonFiberwiththebufferwhileremovingairpoketsfoamthefiberbed.Step2:EquilibrationofNylonFiberPassthewarm15mLMEM,containing5%FCS,at37throughthecolumntoequilibratethefiberwiththeMEM,andthenclosethestopcockvalvetostoptheflow.Step3:ApplicationofCellSuspensioncells,Add2mLof24suspendedinMEMcontaining5%FCSat4,tothecolumn,andthenopenthevalveslowlytoallowthesuspensiontosettleinthefiberbed.Afterthesuspensionsinksthoroughly,thestopcockisclosed.108MEM20mLMEM5%FCS,3715mLCellSuspension24108cellsmLStep4:AbsorptionofcellstothefiberAfteradditionof1mLMEM,containing5%FCS,at37tothecolumn,coverthetopofthecolumnwithaluminiumfoil,andforthenletitincubateat374560minutes.MEM5%FCS,371mLIncubationfor4560min.at37Step5:FractionationandCollectionofTcellsAfterincubation,collectthecellsuspentionelutedfromthecolumnbycarefullyadding20mLMEM,containing5%FCSat37withaflowrateof3-4mL/minute.Additionalpressureotherthangravityshoudnotbeaddedtothecolumnduringtheelution.MEM5%FCS,3720mL3.DeterminationofBcellContaminationAssessBcellcontaminationinobtainedTcellsuspentionbyimmunofluores-cencestainingofthecellusingAntiRatIg,Goatastheprimaryantibodyandfluorescein-labeledAntiGoatIg.4.CapabilityofNylonFiberColumnT(L-Type)CellRecovery:min.25%(Rat)BcellContamination:max.15%ReferencedataCellRecovery:2030%(human),2535%(rabbit)BcellContamination:lessthan5%(human,rabbit)Storageconditions:ProtectfromlightPackage:Column,packedwith1gNylonFiber10Note:NylonFiberColumnT(L-Type)issterilizedbyElectronBeamRadiation.Reference1)Julius,M.H.,Simpson,E.andHerzenberg,R.A.:Eur.J.Immunol.,3,645(1973)WakoPureChemicalIndustries,Ltd.
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