CellularAntioxidantActivityinHepG2Cells

A.SubcultureofCells

Materials:

1.Cellculturemedium:HepG2----CM;MCP-7----DMEM

2.Cellwashmedium:DMEM（2.5%FBS）with10mMHepes,50unites/mL

penicillin,50μg/mLstreptomycin,and100μg/mLgentamicin.

3.Trypsin-EDTA:1X,0.05%,GIBCO,InvitrogenCorporation,Lot:435993

4.TrypanblueStain:0.4%,Lot:347940,GIBCO,InvitrogenCorporation,GrandIsland,NY,USA.

5.PBS

6.BeckmancentrifugeGS-6R

Method:

Coolthetemperatureto4°CinBeckmancentrifuge.

Keepallmedia,PBS,trypsinoniceduringthewholeprocedure.

Removeallgrowthmediafrom6-wellplatesorT75flakes,andwashtwicewith5mLcoldPBS.

RemoveallPBSfromwellsorflasks.

Addtrypsin/EDTAtowellsorflasks(0.6mL/wellfor6wellplates,and2.5mLperT75flask).

Trypsinizequickly–aroundoneminute(1--1.5min)atroomtemperature,ormonitorcellstartingshrinkundermicroscope.

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.

Centrifuge4minutesat650rpmat4°C.

Discardthesupernatantandmixthecellsbytappingthetubewithyourfinger.Whencellsarehomogenous,add40mL2.5%FBS/DMEMandmixwellbyup-down.Centrifuge4minat650rpmat4°C.

Discardsupernatantandmixcellpelletbytappingtubewithfinger.Thecellsareresuspendedin5mLCM(FBS/DMEM)(4°C)andenumerated.(For2x6-wellplatesor2xT75flasks,resupendthecellsin5mL,andkeepcellcounts>20perfield).Keepsuspensiononice.

Add50μLofcellsolutioninto450μLtrypanbluedye.Calculatethecellnumberbycountingatleast6fieldsandaveragingthecounting.

Cellcount:50μLofcellsolution+450μLoftrypanblue

Calculation:Cell#x10x10,000=cells/mL

Totalcellnumber:Cells/mLxtotalvolumeofcellsolution

Calculatetheamountofcellsyouneed.

Dividethenumberofcellsyouneedbythenumberofcellsyouhave=numberofmLofcellsuspensiontouse.(Itiseasiertowith1.0x106cellspermL).

Platecellsandcultureat37°Cin5%CO2.

Afterseeding0.5h,verygentlyrocktheplate/flasktodistributethecellsevenlyonthebottom.

After2.5hincubation,gentlyrockmediuminplate/flaskthreetimesandremovenon-adherentcells.Adherentcellmonolayerswillbeincubatedin5%FBS/DMEMorCM.Writedownthetimeanddateofmediumchange.

Changethemediumnextdaymorning(lessthan20-24h).

Tomaintaincells:1)Checkthecellconditionatleasttwiceaday;2)Changethemediumevery2daysaftertheseconddaymediumchangeuntilplatingagain;3)Changemediumifyouseefloatingcells;and4)Passagecellsbeforereach100%confluence.

B.CAA

Materials

1.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-glutamine

and10mMHepes.

3.Oxidanttreatmentmedium:Hanks’BalancedSaltSolution(HBSS)(nophenol

red)+10mMHepes.

4.20mMstocksolutionofDCFH-DA:inmethanolwasprepared,aliquoted,and

storedat-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=271.17

Workingsolution(600μM):21μLstockaddedto7mLoxidanttreatmentmedium(makefresh).

6.Extractswereobtainedfromthefruitsusing80%acetone.Fruitextractswere

dilutedinantioxidanttreatmentmedium.Finaltreatmentsolutionscontained<2%solvent,andtherewasnocytotoxicitytoHepG2cellsatthoseconcentrations.

7.10mMQuercetinsolution:dissolved33.8mgquercetinin10mLdimethylsulfoxidebeforefurtherdilutioninAntioxidanttreatmentmedium(WMEwith2mML-glutamineand10mMHepes)．

8.Black96-wellplateswithclearflatbottoms

9.12-channelpipetman

10.FluoroskanAscentFLplate-reader

Methods

HepG2cellswereseededat6×104/wellona96-wellplatein100μLofgrowthmedium(CM).Don’tusetheoutsidewells,asthereismuchmorevariationfromthemthantheinnerwells.Seedintriplicatewellsforcontrol,treatments,andblank.Cellsusedinthisstudywerebetweenpassages12and35.

Approximately24hafterseeding,removemediumandwashwith100μLPBSonce.

Treatintriplicatewith100μLofsolutionscontainingdifferentconcentrationantioxidantcompoundsorfruitextractsplus25μMDCFH-DA(finalconcentration)dissolvedinantioxidanttreatmentmediumfor1hat37°C.Solventconcentrationsshouldnotexceed1%.Don’tuseextracts/compoundsatcytotoxicdoses(i.e.,doesthatinhibitcellnumberby>10%after24hasdeterminedbymethyleneblueassay).

Controlwells:Add100μLof25μMDCFH-DA+H2O.

Blankwells:Add100μLof25μMDCFH-DA+H2O.

Removetreatmentmedium.IfaPBSwashistobeutilized,washcellswith100μLPBSonce,otherwise,gotostep5.

Apply100μLoxidanttreatmentmediumcontainingnoABAPtotheblankwells.Apply100μLof600μMABAPinoxidanttreatmentmediumtoallotherwellsusing12-channelpipetmanandplaceinFluoroskanAscentFLplate-reader(37°C)immediately.Measureemissionat538nm(incidentwavelength=485nm)every5minfor1h.

Oneachplateincludecontrolandblankwells.ControlwellscontaincellstreatedwithDCFH-DAandABAP,blankwellscontaincellstreatedwithDCFH-DAandnoABAP.

Afterblanksubtractionfromthefluorescencereadings,theareaunderthecurveoffluorescenceversustimewasintegratedtocalculatetheCAAvalueateachconcentrationofpurephytochemicalcompoundorfruitextractasfollows:

CAAunit=100-(∫SA?∫CA)×100

where∫SAistheintegratedareaunderthesamplefluorescenceversus

timecurveand∫CAistheintegratedareafromthecontrolcurve.Themedianeffectivedose(EC50)wasdeterminedforthepurephytochemicalcompoundsandfruitextractsfromthemedianeffectplotoflog(fa/fu)versuslog(dose),wherefaisthefractionaffectedandfuisthefractionunaffectedbythetreatment.Ineachexperiment,quercetinwasusedasastandard,andcellularantioxidantactivitiesforpurephytochemicalcompoundswereexpressedasmicromolesofquercetin

equivalents(QE)per100μmolofcompound,whereasforfruitextractstheywereexpressedasmicromolesofQEper100goffruit.Tocomparetheantioxidantqualityofdifferentfruits,CAAwasalsocalculatedasmicromolesofQEper100μmoloftotalphenolics.log(fa/fu)=log(CAAunit/(100-CAAunit))

Microplatelayout:Sampleaddedinto96-Well:B,Bla—blank;Cont—Control;S—sample;S1--Sample1,Sii—theIconcentrationofSampleI.

1

2

3

4

5

6

7

8

9

10

11

12

A

B

B

B

B

B

B

B

B

B

B

B

B

B

B

Contr

S11

S12

S13

S14

S15

S16

S31

S32

S33

B

C

B

Contr

S11

S12

S13

S14

S15

S16

S31

S32

S33

B

D

B

Contr

S11

S12

S13

S14

S15

S16

S31

S32

S33

B

E

B

Bla

S21

S22

S23

S24

S25

S26

S34

S35

S36

B

F

B

Bla

S21

S22

S23

S24

S25

S26

S34

S35

S36

B

G

B

Bla

S21

S22

S23

S24

S25

S26

S34

S35

S36

B

H

B

B

B

B

B

B

B

B

B

B

B

B

CAAofBluberry

1.WildBluberryExtract:1.0g/mL

2.DesignedDose(mg/mL)forCAA:

PBSwash:5,10,15,20,30,40

Nowash:1,2,3,4,6,10

Final

concentration

(mg/mL)

Initialconcentration

(mg/mL)

Extractvolume

(μL)

Watervolume

(μL)

WMEvolume

(μL)

1

2

2

98

900

2

4

4

96

900

3

6

6

94

900

4

8

8

92

900

5

10

10

90

900

6

12

12

88

900

10

20

20

80

900

15

30

30

70

900

20

40

40

60

900

30

60

60

40

900

40

80

80

20

900

0

0

0

100

900

CAAofQurcetinStandard

1.10mMquercetinstocksolution:weigh33.8mgquercetinin10mLDMSO.

2.DesignedDose(uM)forCAA:

PBSwash:1,2,4,6,8,10

Nowash:1,2,4,6,8,10

Initialsolution(mM)

Stockvolume(μL)

DMSOvolume(μL)

0.2

2

98

0.4

4

96

0.8

8

92

1.2

12

88

1.6

16

84

2.0

20

80

0

0

100

Treatmentsolution:Add5μLinitialsolution-----495μLWME(Treatmentmedium)-----500μL50μMDCFH-DAintreatmentmedium.

DataTreatmentandCalculations:

ThefollowingrequirestheuseoftheAscentSoftwareversion2.6foundontheFluoroskanAscentFLplatereader,theExcelprogramcalled“CAA–Template”andtheSigmaPlotprogram.BesuretousetheappropriatetabintheExcelprogram;PBSwashornoPBSwash.

SelectalltherawdatafromcellA1inthe“Measures1”tabinAscentSoftwareversion2.6andcopyintoExcelatcellA1.SelectalltherawdatafromcellA1inthe“Blanks1”tabinAscentSoftwareversion2.6andcopyintoExcelatcellO1.

Changethedatalabelsaccordingtotheconcentrationsofquercetinstandard,purecompound,orsampleextractusedinrowsAE3–AJ3,AE20–AJ20,andAE37–AJ37.ThecurvesgeneratedshouldbesimilartothoseseeninFigure2.

CopydatafromBD4–BD16/BD4–BK4andpasteincell1-1inSigmaPlot.

Selectallthedata,choose“ScatterPlot”,then“MultipleScatter”.

Select“XManyY”fromthe“DataFormat”menuandclick“Finish”.

Goto“Tools”“Macro”“Macros”andrunthe“AreaBelowCurves”program.

Clickthe“Compute”buttonthenthe“NextCurve”buttonuntilalltheareaunderthecurveforeachconcentrationofcompound/samplehasbeencalculated.Valueswillbedisplayedincolumns9and10.

Copythevaluesincolumn9andright-click“TransposePaste”thevaluesbelowthedataset.Youshouldendupwithsevenvalues.

Repeatfromstep3forotherreplicatesofthesamesampleandforothersamples.ThesubsequentdatasetscanbepastedoverthepreviousdatasetinSigmaPlot.Subsequentareaunderthecurvevalueswillbedisplayedincolumns11and12(replicate2)and13and14(replicate3).“TransposePaste”theareaunderthecurvevaluesdirectlybelowthatoftheprevioussample.

SelectthethreesetsofareaunderthecurvevaluesandpasteintotheExcel