LacrimalExperimentalProtocol(4/4/94)

DayBefore:

1.Prepare(6)50mlconicalseachcontaining50mlofDMEM(highglucose)andgentamycin(50µg/ml);placeinincubator(90%O2,10%CO2).

2.ThawFBS(placeat4°C).

3.PlaceBMSat4°CandLMat4°C.Ifnotplanningtousegelledsubstrate,coatat4-8µg/wellin100µlofdH2Oovernightat4°C.

DayofExperiment:

1.ThawoniceSTI,hyaluronidase,Collagenase,DNase,dexamethasone,putrescine,ITS,glutathione,EGF

2.Weighout12.5mgofascorbicacid(to1mlwithddH2Olater,thenfiltersterilize).

3.Makeup1mlofSTIin100mlofDMEM/gent.(doas(2)50ml);mixwellandadd5mleachtosix60mmdishes;placeinincubator.

4.Makeenzymesolutionmixingtogether500µlofhyaluronidase(Worthington"HSEP";stock6980U/ml),500µlofcollagenase(Worthington;1:1mixtureoftypesIIandIV;stock4000U/ml),and100µlofDNase(stock1000U/ml)to8.4mlofDMEM/gent.

5.Prepare40mlofEDTA/HBSSconsistingof:4mlof10xHBSS,8mlof10xEDTA,and28mloftissueculturesterilewater.

6.MakeupDMEM/gent.plusgoodiesasfollows(forSFMOM,deleteFBS,increaseDMEM/gent.to28.5mlandadd30µlof10µg/mlbFGF(SigmaF9786):dexamethasone30µlof10µg/mlstockputrescine300µlof100mMstock[l-ascorbicacid60µlof12.5mg/mlstock]*ITS360µlof1.25mg/ml;1.25µg/mlstockglutathione300µlof1mg/mlstockEGF30µlof50µg/mlstockHEPES456µlof1MstockFBS3ml(heatdeactivated)DMEM/gent.25.5ml(highglucose)*addjustbeforeuse7.PrepareDMEM/STI/20%FBSbymixing40mlofDMEM/STI/gent.with10mlofFBS.

8.Perfuse(3)male4wkSpragueDawleyrats(fromNCI)withDMEM/gent.(have100mlmadeup)for3-5minuntillungandliverarewhite.

9.Removelacrimalglandsandplaceina60mmdishcontainingicecoldDMEM/STI/gent.Placedishonicewhilesecondanimalisdissected.

10.Inhood,transfertonewDMEM/STI/gent,andcutawayductwithscissors.

11.Transfertoanewdishandstab(25guageneedle)andinjecteachglandtwotimeswith1mlofmedia.Useneedlestopullapartgland.Trytoremovecapsule.

12.Minceglands(1-4mm2pieces)withtwo#10bladescapels,thentransfermincedpiecesintoanautoclaved25mlplasticflaskusingatransferpipet.RinsedishwithDMEM/STI/gent.

13.Allowpiecestosettle,thenpulloffmediumandadd3transferpipetvolumesofHBSS/EDTA/gent.

14.Letsettle,thendrawoffandadd8mlofHBSS/EDTA/gent.Puttoponflaskandplaceinincubatoronvortexatlowestsetting(120oscillations/min)for15min.

15.Coatwellsofa96wellplatewith0.18mg/wellofBMS(=40µl/wellof#634[10mg/ml]diluted180µlupto400µlwithcoldDMEM;tubeonice;pre-chilledpipettip);or106µl/wellof#754laminin[1.7mg/ml].Forcoating,plateisplacedonice,thenisgelledat37°Cinincubatorfor1hr.

16.Letsettle,removesupernatantandadd7mlofDMEM/STI/gent.for2min.

17.RemoveDMEM/STI/gent.andadd5mlofenzymemixtureinDMEM/gent.Placeonvortexfor15minat37°C.

18.AdjustPercollbyadding1mlof10xsaline(1.5MNaCl)to9mlofPercoll.Makeup10%(0.33mladjustedPercoll+2.67mlDMEM/STI/20%FBS),30%(1mladjustedPercoll+2mlDMEM/STI/20%FBS)and60%(2mladjustedPercoll+1mlDMEM/STI/20%FBS)Percoll.Poura60%/30%/10%grADIentusinga1mlpipet.Placeinincubator.

19.Transfersolutiontoa15mltube.RinseoutflaskwithDMEM/STI/gent.andaddwashto15mltube.Do2washes.

20.Spincellsat500rpmfor3min(RT).

21.Discardsupernatant,flickbrieflytoloosenpelletandaddHBSS/EDTA/gent.to12ml.Inverttomixandspinfor3minat500rpm.

22.Discardsupernatant.Add3mlofHBSS/EDTA/gent.topellet.Loosenpelletbygentlydrawinginandoutwithatransferpipet;transfertoflask.Washouttubewith2mlofHBSS/EDTA/gent.;transfertoflaskandmakeupto8ml.

23.Incubateat37°Convortexfor15min.

24.Ininhibitionexperiments,add50µg/wellantibodytoBMSfor1hrat37°C.

25.Transferto15mlconical.WashflaskouttwotimeswithDMEM/STI/gentandspinat500rpmfor3min.

26.Discardsupernatant,loosenpelletandadd2mlofenzymesolution.Transfertoflaskandwashouttubewith3mlofenzymesolution.Incubateflaskonvortexat37°Cfor20min.

27.RemovesUSPensionandplaceina15mltube.Washoutflaskwith20%FBS/DMEM/STI/gent.andmixbyinversion2-3x.Spinat500rpmfor3min.

28.Discardsupernatantandsuspendin10mlof20%FBS/DMEM/STI/gent.Transfertoa50mlconical.Use10mlsyringetoremovecells;removeneedle.

28.Filterthrough160µmand25µmNitexfiltershookedupintandem;filterintoa15mltube.Washwith3mlof20%FBS/DMEM/STI/gent.Spinat500rpmfor3min,thenresuspendpelletin6mlof20%FBS/DMEM/STI/gent.

29.Addveryslowlytotopof60/30/10%Percollgradientandspinat500rpmfor15min.

30.Ifdoingantibodyinhibitionexperiments,removeantibodyandwashBMStwotimeswithwarmDMEM/gent(gel)orsterilePBS(coatedwells).

31.Cellsresideat60/30%interface.UsetransferpipettopulloffPercolltothisinterface,thenpulloffcellsandresuspendin20%FBS/DMEM/STI/gent.Spinat500rpmfor3min.Resuspendagainin20%FBS/DMEM/STI/gent.(14ml)andspintoremoveallPercoll.

32.Discardsupernatantandmakeupin8mlofDMEM/gent.plusgoodies.Take15µlofcellsuspensionplus15µloftrypanblue,mixgentlythenadd15µltoeachsideofthehemocytometer.Count#ofdead(blue)andlivingcellsingridusing10xobjectivelens.Cellnumberinsuspensionis[2(Y)x104/ml],whereYisnumberofcellsingrid.

33.AddDMEM/gent.plusgoodies,suchthathave6x105cells/ml.Platecellswithmultipipettorat91µl/well(0.55x105cells/well).Incubateovernightin6%CO2incubator.

NextDay

1.Placedispaseandtrypsinonice.

2.Letcarbacholwarmuponbench.

3.Makeup6mloffreshDMEM/gent.plusgoodiesusingDMEM/gent.fromincubator.

4.Carefullypulloffmediafromovernightcultureandretainforcellcount.Washtwotimeswith100µleachofDMEM/gent.plusgoodies(addwithmultipipettor.Add75µlofDMEM/gent.plusgoodiesandincubatefor100min.

5.Weighout5.5mgofcarbachol.PutVIPstockonice.LabeltwosetsofT=0tubesandtwosetsofT=100mintubes.

6.15minbeforeendofaboveincubation,makeupstimulationmedium.Make10µlofVIPstockto100µlofDMEMtogive10-6MVIP.Make5.5mgofcarbacholupin3mlofDMEMtogive10-2Mcarbachol.Add60µlof10-6MVIPand60µlof10-2Mcarbacholto6mlofDMEM/gent.plusgoodiestogivestimulationmedium.

7.Removeandretainmedia(ÔT=0minÕ).Replacewith75µlofstimulationmediumusingmultipipettor.Incubatefor100minat37°C.

8.SpinT=0mediaatsetting6for6minonEppendorfcentrifuge.Retainsupernatantandstoreat-70°C.Makeuptrypsin/EDTA/dispasebyadding0.9mlof10xtrypsin/EDTAto3.6mlofdispase;putat37°C

9.After100min,removeandretainstimulationmedia.Add100µl/wellofdispase/trypsin.Placeplateonvortexat37°Cfor30-45min.

10.Spinstimulationmediumatsetting6for6minonEpendorf,retainsupernatantasT=100andstoreat-70°C.Keeppelletonicetocombinewithdispase/trypsinremovedcells.

11.Loosencellsandpulloffdispase/trypsincellsuspensionbydrawingupanddowninpipettipandplaceintubecontainingcellpelletfromabove.Washeachwellonetimewith100µlofDMEMandplaceincellpellettube.Spinatsetting6for6minonEpendorfcentrifuge.Pulloffsupernatantwithaspiratorandadd121.2µlofDMEM.Vortexandstoreat-70°C.

AssayofSamples

A.PeroxidaseAssay

1.Putmediasamplesat4°Ctothaw.Cellpelletshouldbequicklyrefrozenandreplacedat4°C.PullDABfromfreezertowarmup.Put1U/µlperoxidasestockonice.

2.Weighout0.1gofDAB;placeindark.

3.Make2.7mlof1MTris,pH8,to20mlgiving0.136MTris.

4.Add5.4µlof30%H2O2to6mlofwater,andstoreonice.

5.Turnoncomputerandsetuptemplate.Usekinetic(L1),automixon,wavelength450nm,runtime5:00(5min),readinterval0:10(10sec).

6.Foraperoxidasestandardcurve,make10µlofperoxidasestockupto1000µlinDMEMtogiveasolutionof10mU/µl.Make100µlof10mU/µlupto1000µltogive1mU/µl.Make100µlof1mU/µlto1000µltogive0.1mU/µl.

7.To96wellplate(onice)setupseveralblanks,astandardcurveandT=0,T=100andcellsupernatantusingSoftmax.Forblanks,use50µlofDMEM.Forunknownsuse50µl.Setupstandardcurveasfollows(haveazeropoint[STD01]):STD025mµ0µlDMEM50µlof0.1mµ/mlSTD0310mµ40µlDMEM10µlof1mµ/mlSTD0450mµ0µlDMEM50µlof1mµ/mlSTD05100mµ40µlDMEM10µlof10mµ/ml8.AddTristoDABandvortextodissolve.AdjustpHto7.0withabout10dropsof1NNaOH.Drawupwithasyringeandpassthrougha.2µmfilterintoareagentreservoir.

9.Toblanks,standardsorunknowns,userepeatorpipet(setting5)toadd125µl/wellofDABsolution.PlaceplateonUVmaxdrawer,thenwithfreshtipsandreservoiruserepeatorpipet(setting1)toadd25µl/wellofH2O2solution,andstartreadingimmediatelyfor2minwithautomixat450nm.Afterrun,resetVmax(usually5-7points).

10.PlotandresetVmaxtorepresentsteepestpartofcurve(5-10points).Slopeforeachstandardcurveshouldbesimilar.

B.DNAAssay

1.MakeupCapillaryAssaySolutionconsistingof400µlof2.5xmodifiedTNE,20µlofHoechstdye(1mg/ml)and580µloffiltereddH2O.[For2.5xmodifiedTNE,mixinaconical:40mlof5MNaCl,1.25mlof1MTrispH8,0.25mlof0.5MEDTA,pHto7.4with5-7dropsof1MHCl,thenmakeupto50mlwith5MNaClandpassthrougha0.2µmfilter;forHoechstdye,dissolve10mgofdyein10mlofmolecularBIOLOGygradedH2Oandpassthrougha0.2µmfilter;preparealsofilteredmolecularbiologygradedH2O;store2.5xTNE,HoechstdyeanddH2Otogetherinacoveredboxat4°C].

2.MakeupcalfthymusDNAstandardsolutions:[A]10µlof1mg/mlstockmadeupto1000µlinDMEM,[B]30µlof"A"madeupto100µlinDMEM,[C]20µlof"A"madeupto100µlofDMEM,[D]10µlof"A"madeupto100µlDMEM.Use50µlof"D"for50ng,50µlof"C"for100ng,50µlof"B"for150ng,and50µlof"A"for500ng.Spin10minatRT.

3.Inependorftube,mixtogether40µlof2.5xmod.TNE,12.5µlofspunsampleplus37.5µlDMEM,(or50µlstandardmadeupabove),and10µlofcapillaryassaysolution,mix.Storetemporarilyindark.Forblankuse50µlDMEMplusTNEandcapillaryassaysolution.Vortex,thenspin2minatRT.

4.Add100µlofblanktoendof100µlcapillarytube,keeptubehorizontalandsealwithputtymaterial.Insertoppositeendoftubeintoholder,andthenplaceinminifluorometer.Zerowithzeroknob.

5.Insertcapillarytubecontaining100ngofDNAandusescaleknobtosetto25(readsas.25unitsperngDNA).Insertandreadotherstandardsandunknowns.Makesurenofingerprintsoncapillarytube.FinalDNAvalueisreadingx16(4xfrominstrumentstandardization;4xfromdilutionfactor).

6.CombinedatafromA.andB.inExcel.Openanewworksheet.Typetestsubstrates(ie.BMSorLn)foreachof96wellsincolumnA.Type"T=0"attopofcolumnB,followedbyVmaxvalues(startatB2).Dothesamefor"T=100"(columnC),"NotSecreted"(columnD)and"Total(B+C+D)"(columnE).ToaddB+C+D,typeinE2"=B2+C2+D2"andhitreturn.Toapplythiscalculationtoallotherwells,clickonE2andblockoutEcolumn,undereditgoto"filldown"andthenhitreturn.TonormalizetoDNAvalues,copyandpasteallofcolumnAnamestoalowerportionofcolumnA(ieA40).InB40,typeintheµgDNAvalueforA2(obtainedby16xfluorometerreading).FillinbelowallotherDNAvaluesandtitlecolumn"µgDNA".Titlethreeadjacentcolumns"T0(mU/µgDNA)",T100(mU/µgDNA)",and"Tot(mU/µgDNA),"respectively.Columnscanbewidenedbydraggingcolumnboundarytoright.ToobtainnormalizedsecretionvaluesinmU/µgDNA,typeinC40"=(B2*0.3165)/B40"andhitreturn.ToapplytoallofcolumnC,clickinC40,blockoutcolumn,undereditgoto"filldown"andhitreturn.ForcolumnD,useformula"=(D2*0.3165)/B40"andforcolumnE"=(E2*0.3165)/B40".

C.MTTAssay

1.Coat96wellplatewith1/3theamountBMSusedon48wellplate.

2.Add1/3theamountofcellsusedona48wellplatein1/3thevolume.

3.Incubatecellsforaslongasexperimentrequires(overnightusually).Addreagentstobetestedtothewell(again1/3theamountin1/3thevolume).

4.Afterincubationfortimerequiredbyparticularexperiment,addenoughmediatothewellstomakethefinalvolume100µl.

5.Add10µlofMTTA-BSolution.Mixwellbytappingsidesofplate.Incubatefor4hours.

6.Add100µlofMTTSolutionCtoeachwell.Mixthoroughlybyrepeatedpipettingwithamultichannelpipettoruntiltheblack,fuzzycrystalsonthebottomofthewellhavedissolved.

7.Within1hourmeasure,theabsorbanceonanELISAplatereader.Useatestwavelengthof595nmandareferencewavelengthof630nm.

D.LDHAssay

1.SetupUVmaxtoreadkineticallyfor5minwithautomixat490nm.

2.Dilute2mlof1MTris,pH8,in10mlofddH2Oandadd49mgofL(+)lactate.

3.On96wellplatealiquot50µloft=0,t=100orcellpelletsupernatant.Forblank,useDMEM(forcellpellet)andDMEM+goodies(fort=0andt=100;sinceDMEM+goodiesgivesrisetoachangeinreactionproduct).Use25µl"multi-enzymelin-trol"(Sigma#M2266)dilutedwith25µlofDMEMaspositivecontrol.

4.Add12.8mgofadrypremixofINT(Sigma#I-8377),PMS(Sigma#P9625)andNAD(Sigma#N-7004)toTris/lactatesolution.Mixandwrapinfoil.(Premixis167mgofINT,43mgofPMSand431mgofNAD.)

5.Place96wellplateinUVmaxandadd125µl/wellofTris/lactate/INT/PMS/NAD.

6.Expressvaluesas%oftotal.

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