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AIM-V®MediumCTS™TherapeuticGradeserumfreecellexpansionmediumGIBCO®AIM-VMediumCTS™(TherapeuticGrade)isthefirstcommerciallyavailabledefined,serum-freeformulationforproliferationand/ormanipulationofT-cellsanddendriticcellsandmanufacturedincompliancewithcGMP.AIM-VMediumCTS™isanFDA510(k)cleareddevicewhichisintendedforhumanex-vivotissue&cellcultureprocessingapplications.DescriptionCat.No.SizeAIM-V®MediumCTS™,Liquid0870112DK1000mLAIM-V®MediumCTS™,Liquid0870112BK10L(Bag)IntendedUseForhumanex-vivotissue&cellcultureprocessingapplications.CAUTION:Whenusedasamedicaldevice,FederalLawrestrictsthisdevicetosalebyorontheorderofaphysician.StorageStoremediumat2to8°C.Protectfromlight.ShelfLife14monthsCultureProcedure:TheprocedurebelowservesasageneralguidelineforstaticT-cellanddendriticcellculture,regardlessofvessel.Forhigh-densitycultureinbioreactors,optimalproceduresshouldbedeterminedempiricallybytheinvestigator.TCellsCulture:1.Preparefreshperipheralbloodmononuclearcells(PBMCs)orrapidlythaw(1minute)frozenvialsofPBMCscellsina37°CwaterbathaccordingtostandardPBMCthawingprotocols.2.WashcellswithDPBSCTS™withoutcalciumandmagnesium(Cat.NoA12856),with2-5%heat-inactivatedhumanpooledTypeABserumaccordingtotheapplications,ifdesiredorrequired.3.Countcellsusingeitherelectronic(i.e.CoulterCounter,Vi-Cell)ormanual(i.e.hemocytometer)methods.4.Centrifugecellsandremovewashbuffer.5.ResuspendPBMCatroughly0.5-1x106CD3+Tcells/mLinmediumsupplementedwithcytokines(e.g.IL-2),ifusedatcultureinitiation.Transferthedesirednumberofcellstothedesiredtissueculturevessel.AvarietyofprotocolsmaybeusedforactivatingT-cellsforsubsequentexpansion,includingaddingstimulatoryantibodiesorantigenpresentingcells.Similarly,foreithersmallorthelargescaleT-cellexpansion,cellscanbeisolated,activatedandexpandedwithDynabeads®ClinExVivo™CD3/CD28orDynabeads®CD3/CD28CTSTM(Cat.No.402-03D)accordingtoinstructionsintheproductinsert.6.Incubatetheculturevesselat37°Cinahumidifiedatmospherewith5%CO2.Feedandmaintaincellsatdesiredconcentrationswhilecellsareinlogphasegrowth.Tomaintainlogphasegrowth,itmaybepreferabletosplitcellstoachieveadensityof0.5-1x106cells/mLwhenevercelldensitygetsabove1x106cells/mL(e.g.2x106cells/mL,split1:4tocontinuecultureat0.5x106cells/mL).Foroptimalgasexchangeinstaticplateculturesitisrecommendedthatmediumdepthnotexceed1to1.2cm.MonocyteDerivedDendriticCellCulture:1.Preparefreshperipheralbloodmononuclearcells(PBMCs).2.PlatePBMCincultureflaskwith25mLRPMI1640(Cat.No72400)orAIM-V®MediumCTS™(TherapeuticGrade).3.Incubatefor2to3hoursat36to38˚Cinahumidifiedatmosphereof5%CO2inair.4.Discardmediumcontainingnon-adherentcells.5.Washtheadherentcells(mainlyCD14+monocytes)threetimeswithDPBSwithoutcalciumandmagnesium(Cat.NoA12856).6.Addmediumcontaining50to100ng/mLrecombinanthumanIL-4(Cat.No.CTP00431mgorCat.No.CTP0041100ug)and50ng/mLrecombinanthumanGM-CSF(Cat.No.CTP2011100ugorCat.No.CTP20131mg).Celldensityshouldbebetween1to3x105cells/mL.7.Incubatecellsat36to38˚Cinahumidifiedatmosphereof5%CO2inairfor5days.Itisrecommendedtoreplacemediumonceafter3dayswithfreshmediumcontainingIL-4andGM-CSF.Saveallnon-adherentorlooselyadherentcellsbycentrifugingtheremovedculturemedium10minutesat200xgandaddingthepellettothefreshculturemedium.8.After6days,thelooselyadherentornon-adherentcellsshoulddisplaytypicaldendriticcellmorphologyandsurfacemarkers(CD1a,CD80,CD86,andHLA-DR).9.Thematurationofdendriticcellsisinducedbytheadditionofeither1µg/mLLPSor50µl/mLTNF-α(cat.No.CTP30131mgorCat.No.CTP3011100ug)tothemedium.Note:Alternativelytoplasticadherence,monocytescanalsobeisolatedbymagneticseparation.RelatedProductsDulbecco'sPhosphateBufferedSalineCTS™(DPBS)withoutcalcium,magnesium(1X),liquid(A12856)L-Glutamine-200mM(100X),liquid(25030)DynabeadsClinExVivo™CD3/CD28®orDynabeadsCD3/CD28®CTSTM(402-03D)DynaMag™CTS™(121-02)IL-2CTS™RECHU(CTP0021100ugorCTP00231mg)IL-7CTS™RECHU(CTP0071100ugorCTP00731mg)IL-4CTS™RECHU(CTP0041100ugorCTP00431mg)GM-CSFCTS™RECHU(CTP2011100ugorCTP20131mg)TNF-αCTS™(CTP3011100ugorCTP30131mg)TechnicalSupportForadditionalproductandtechnicalinformation,suchasMaterialSafetyDataSheets(MSDS),CertificateofAnalysis,etc,pleasevisitourwebsiteat@lifetech.comThetrademarksmentionedhereinarethepropertyofLifeTechnologiesCorporationortheirrespectiveownersReferences1.RebeccaJetal.,(2010)NaturalexposuretocutaneousanthraxgiveslonglastingTcellimmunityencompassinginfection-specificEpitopes.J.Immunol.,184:3814–38212.FabriciusDetal.,(2010)ProstaglandinE2inhibitsIFN-αsecretionandTh1costimulationbyhumanplasmacytoiddendriticcellsviaE-prostanoid2andE-prostanoid4receptorengagement.J.Immunol.,184:677–6843.NesbitLetal.,(2010)PolyfunctionalTLymphocytesAreinthePeripheralBloodofDonorsNaturallyImmunetoCoccidioidomycosisandAreNotInducedbyDendriticCells.Infect.Immun.,78:309-3154.JahrsdorferBetal.,(2010)GranzymeBproducedbyhumanplasmacytoiddendriticcellssuppressesT-cellexpansion.Blood,115:1156–11655.CsillagAetal.,(2010)