Introduction Obesityisasignificantclinicalproblemthatcontributestolife-threateningdiseasessuchasdiabetesandatherosclerosis.Withanincreasingincidenceofobesityworldwide,rationalstrategiesareneededtocontrolADIpogenesis.Cellsthatundergodeterminationtotheadiposelineagecalledadipoblastsandthisprocesscalledadipogenesis.Adipogenesishasbeenextensivelystudiedusinginvitromodelsystemsconsistingeitherofestablishedadipogeniccelllines(clonallines3T3-L1,3T3-F442A,Ob17,BFC-1,ST13,A31T,...)andprimarycultureofadipocyteprecursorsandpre-adipocytes.GrowtharrestofadipoblastsattheG1/SphaseofthecellcycleappearstoberequiredforthecommitmentofadipoblaststopreadiposecellswhichisaccompaniedbytheemergenceofearlyMarkersofadipgenesis.Usingcellculturesystemscultivatedeitherinthepresenceofserumorindefinedmedium,ithasbeenpossIBLetoidentifyfactorsregulatingeitherpositivelyornegativelyadiposedifferentiation.Thepositiveregulatorsincludeseveralhormonessuchasinsulin,growthhormone,andtriiodothronine.Inaddition,serum,partiallypurifiedserumfactors,andconditionedmediumfromseveraladipogeniccelllineshavebeenshowntostimulateadiposedifferentiationinvitroandassumedtocontainadipogenicregulatorsdistinctfromthehormones.Mammalian3T3-L1cellsdifferentiateintoadipocytesaftercontinuousexposuretopharmacologicaldosesofinsulinorphysiologicaldosesofinsulin-likegrowthfactorI. Principle Whenadipoblaststreatedwithacombinationofdexamethasone,isobutylmethylxanthine(IBMXorMIX)andinsulin,cellsstarttoadoptaroundedphenotypeandwithin5-8daysbegintoaccumulatelipidsintracellularlyintheformoflipiddroplets.Theinductioncondtionsandmediavaryaccordingtothecelllines.TreatmentofcellswithdexamethasoneactivatesthetranscriptionfactorCCAAT/enhancer-bindingproteinb(C/EBPb).IBMXinhibitssolublecyclicnucleotidephosphodiesterasesandresultsinincreasedintracellularcAMPlevels.Atthenuclearlevel,treatmentwithIBMXresultsinactivationoftherelatedtranscriptionfactorC/EBPd.C/EBPbanddinturninducetranscriptionofC/EBPaandPPAR.Within3daysofexposuretoinducers,thecellsundergotworoundsofmitosis,termedmitoticclonalexpansion,whicharerequiredfordifferentiation.Insulinorinsulin-likegrowthfactor-1promoteadipocytedifferentiationbyactivatingPI3-kinaseandAktactivity.ModulationoftheactivityoftheforkheadtranscriptionfactorFoxo1appearstobenecessaryforinsulintopromoteadipocytedifferentiation.C/EBPaandPPARdirectthefinalphaseofadipogenesisbyactivatingexpressionofadipocyte-specificgenes,suchasfattyacidsynthetase,fattyacidbindingprotein,leptinandadiponectin. Reagentsandrequirements Materialandsources: Preparationofsolutions Procedures Preadipocytemaintenanceandpassage: Platethecellsin10%CS/FBS-DMEMonculturedishesandincubatethemat37°Cin10%CO2.Itisimportanttofeedthepreadipocyteseverycoupleofdaysandavoidlettingthemgettooconfluent(>70%),ifyouwanttocontinuetopassagethemanddifferentiatethematalaterdate.So,takecaretosplitthemappropriately.Theycanbesplitasfaras1:15,thoughweusuallydo1:10orlessdependingonneed. AdipocyteDifferentiationProtocol Stainingprocedure: Precautions: