细胞抗氧化方法CAA步骤-康奈尔大学刘瑞海实验室实验方法

CellularAntioxidantActivityinHepG2CellsA.SubcultureofCellsMaterials:1.Cellculturemedium:HepG2----CM;MCP-7----DMEM2.Cellwashmedium:DMEM（2.5%FBS）with10mMHepes,50unites/mLpenicillin,50μg/mLstreptomycin,and100μg/mLgentamicin.3.Trypsin-EDTA:1X,0.05%,GIBCO,InvitrogenCorporation,Lot:4359934.TrypanblueStain:0.4%,Lot:347940,GIBCO,InvitrogenCorporation,GrandIsland,NY,USA.5.PBS6.BeckmancentrifugeGS-6RMethod:1.Coolthetemperatureto4°CinBeckmancentrifuge.2.Keepallmedia,PBS,trypsinoniceduringthewholeprocedure.3.Removeallgrowthmediafrom6-wellplatesorT75flakes,andwashtwicewith5mLcoldPBS.4.RemoveallPBSfromwellsorflasks.5.Addtrypsin/EDTAtowellsorflasks(0.6mL/wellfor6wellplates,and2.5mLperT75flask).Trypsinizequickly–aroundoneminute(1--1.5min)atroomtemperature,ormonitorcellstartingshrinkundermicroscope.6.Dislodgecells:1）6wellplate:usingP1000pipetmantodislodgecellsbyup-downblowing.Itiseasiertodislodgecellsbypoolingthetrypsinsolutionfromtwowells(1mLperwell)andrinseoncewiththetrypsinsolutionfromanothertwowells.Thepurposeofup-downblowingistoobtainsinglecells.2)T75flask:Gentlyclappingtheflaskonsidewithyourhandstodislodgecellsandthenusing5-mLpipettodislodgecellsbyup-downblowing.Itiseasiertodislodgecellsbypoolingthetrypsinsolutionfromtwoflask(around5mL).Rinseoncewithwashingmedium.3)Removingthecellsintoa50mLcentrifugetubecontainingaround20mLofcoldwashingmedia(2.5%FBS/DMEM)4)Washthewellsorflaskwith2.5%FBS/DMEM(cold)andtocentrifugetubetoatotalvolumeof35-40mL.Mixwellbyup-down3times.7.Centrifuge4minutesat650rpmat4°C.8.Discardthesupernatantandmixthecellsbytappingthetubewithyourfinger.Whencellsarehomogenous,add40mL2.5%FBS/DMEMandmixwellbyup-down.Centrifuge4minat650rpmat4°C.9.Discardsupernatantandmixcellpelletbytappingtubewithfinger.Thecellsareresuspendedin5mLCM(FBS/DMEM)(4°C)andenumerated.(For2x6-wellplatesor2xT75flasks,resupendthecellsin5mL,andkeepcellcounts20perfield).Keepsuspensiononice.10.Add50μLofcellsolutioninto450μLtrypanbluedye.Calculatethecellnumberbycountingatleast6fieldsandaveragingthecounting.Cellcount:50μLofcellsolution+450μLoftrypanblueCalculation:Cell#x10x10,000=cells/mLTotalcellnumber:Cells/mLxtotalvolumeofcellsolution11.Calculatetheamountofcellsyouneed.Dividethenumberofcellsyouneedbythenumberofcellsyouhave=numberofmLofcellsuspensiontouse.(Itiseasiertowith1.0x106cellspermL).12.Platecellsandcultureat37°Cin5%CO2.13.Afterseeding0.5h,verygentlyrocktheplate/flasktodistributethecellsevenlyonthebottom.14.After2.5hincubation,gentlyrockmediuminplate/flaskthreetimesandremovenon-adherentcells.Adherentcellmonolayerswillbeincubatedin5%FBS/DMEMorCM.Writedownthetimeanddateofmediumchange.15.Changethemediumnextdaymorning(lessthan20-24h).16.Tomaintaincells:1)Checkthecellconditionatleasttwiceaday;2)Changethemediumevery2daysaftertheseconddaymediumchangeuntilplatingagain;3)Changemediumifyouseefloatingcells;and4)Passagecellsbeforereach100%confluence.B.CAAMaterials1.CM:CompleteMedium(William’sMediumE(WME)+5%FBS+10mMHepes+2mML-glutamine+5μg/mLinsulin,0.05μg/mLhydrocortisone,50units/mLpenicillin,50μg/mLstreptomycin,and100μg/mLgentamicin).WME:1X,-L-Glutamine,Invitrogen.2.Antioxidanttreatmentmedium:WMEsupplementedwith2mML-glutamineand10mMHepes.3.Oxidanttreatmentmedium:Hanks’BalancedSaltSolution(HBSS)(nophenolred)+10mMHepes.4.20mMstocksolutionofDCFH-DA:inmethanolwasprepared,aliquoted,andstoredat-20°C.DCFH-DA:2′,7′-dichlorofluorescindiacetate,SigmaD6883.Workingsolution(50μM):e.g.,50μLstocksolutionaddedto20mLantioxidanttreatmentmedium.Combine1:1withantioxidantsolutionsforfinal25μMsolution(makefresh).5.200mMABAPstocksolution,storedat-40°C.Dissolve0.5423gin10mLH2O(aliquotsstoredat-40°C).ABAP:2,2′-azobis(2-amidinopropane)dihydrochloride,WakoChemicals992-11062.FW=271.17Workingsolution(600μM):21μLstockaddedto7mLoxidanttreatmentmedium(makefresh).6.Extractswereobtainedfromthefruitsusing80%acetone.Fruitextractsweredilutedinantioxidanttreatmentmedium.Finaltreatmentsolutionscontained2%solvent,andtherewasnocytotoxicitytoHepG2cellsatthoseconcentrations.7.10mMQuercetinsolution:dissolved33.8mgquercetinin10mLdimethylsulfoxidebeforefurtherdilutioninAntioxidanttreatmentmedium(WMEwith2mML-glutamineand10mMHepes)．8.Black96-wellplateswithclearflatbottoms9.12-channelpipetman10.FluoroskanAscentFLplate-readerMethods1.HepG2cellswereseededat6×104/wellona96-wellplatein100μLofgrowthmedium(CM).Don’tusetheoutsidewells,asthereismuchmorevariationfromthemthantheinnerwells.Seedintriplicatewellsforcontrol,treatments,andblank.Cellsusedinthisstudywerebetweenpassages12and35.2.Approximately24hafterseeding,removemediumandwashwith100μLPBSonce.3.Treatintriplicatewith100μLofsolutionscontainingdifferentconcentrationantioxidantcompoundsorfruitextractsplus25μMDCFH-DA(finalconcentration)dissolvedinantioxidanttreatmentmediumfor1hat37°C.Solventconcentrationsshouldnotexceed1%.Don’tuseextracts/compoundsatcytotoxicdoses(i.e.,doesthatinhibitcellnumberby10%after24hasdeterminedbymethyleneblueassay).Controlwells:Add100μLof25μMDCFH-DA+H2O.Blankwells:Add100μLof25μMDCFH-DA+H2O.4.Removetreatmentmedium.IfaPBSwas