THESEINSTRUCTIONSAPPLYTOORDERSCONTAININGTHEFOLLOWINGCELLPRODUCTS CryopreservedCells(Singledonor) ProliferatingCells Pleasereadandfollowtheseinstructionscarefullyandcompletely.BioWhittakerisnotresponsIBLeforproductlossduetoimproperreceiptandhandlingofitsproductsbycustomers.Replacementproductwillbesentatthecustomer"sexpense. *AA-1005*AA-1005-1Rev.04/98 FibroblastCellSystem 1.NormalHumanDermalFibroblastsandNormalHumanLungFibroblastsfromsingledonors,aseither: Adult ProliferatingT-25FlaskCC-2611 ProliferatingT-75FlaskCC-0252 Proliferating96-wellPlatesCC-0160 CC-3132 Neonatal ProliferatingT-25FlaskCC-2609 ProliferatingT-75FlaskCC-0210 Proliferating96-wellPlatesCC-0116 CC-3132 Adult ProliferatingT-25FlaskCC-2612 ProliferatingT-75FlaskCC-0282 Proliferating96-wellPlatesCC-0164 CC-3132 Theproliferatingculturesareshippedinflasksorplatesfilledwithmedium.Thecellsshouldbebetween30and100%confluentuponarrival.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesQCperformanceresultsanddonorinformation. Thecryopreservedculturesareshippedinascrewcapcryovialcontainingapproximately500,000cells.ACertificateofAnalysisisprovidedwitheachcellstrainandindicatesdateofcryopreservation,QCperformanceresults,donorinformationandthenumberofcellscontainedinthecryovial. FibroblastGrowthMediumBulletKit®(FGM®BulletKit®)(CC-3130),whichcontainsa500mlbottleofFibroblastBasalMedium(FBM®)andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®(amountsindicateconcentrationofeachSingleQuot®) 1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml FibroblastGrowthMedium-2BulletKit®(FGM®-2BulletKit®)(CC-3132),whichcontainsa500mlbottleofFibroblastBasalMedium(FBM®)andallthesupplementslistedbelow,convenientlypackagedassingle-usealiquotscalledSingleQuots®(amountsindicateconcentrationofeachSingleQuot®) 1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml10mlFBS(FetalBovineSerum)(CC-4101) 3.ReagentPackTM(CC-5034)containsone100mlbottleofeachofthefollowingsubculturereagents: HEPESBufferedSalineSolution(HEPES-BSS)(CC-5022)1x100mlbottleTrypsin/EDTASolution(T/E)(CC-5012)1x100mlbottleTrypsinNeutralizingSolution(TNS)(CC-5002)1x100mlbottle NOTE:IfyouuseadifferentClonetics®medium,seeAppendixA. ProductApplicationsClonetics®NormalHumanNHDFandNHLFare: MaterialsNotProvidedFibroblastCellSystemsdonotcontainplasticware,glasswareorotherlaboratoryequipmentusedinacellculturelaboratory.Individualcomponentsareavailableseparately. ProductWarrantyCULTURESHAVEAFINITELifespanINVITRO.BioWhittakerwarrantsitsClonetics®cellsinthefollowingmanneronlyifClonetics®mediaandreagentsareused. CellIsolationFibroblastculturesareestablishedatBioWhittaker"scellculturefacilityfromnormalhumantissue. IfthawedandwillNOTbeusedwithin72hours,growthfactorsmustberefrozen.Theymayberefrozenonlyonceandthenstoredat-20·Cforuptooneyear. StoreFBM®andFBM®-2at2·to8·CStorefullysupplementedFGM®andFGM®-2at2·to8·C. Avoidrepeatedwarmingandcoolingofthemedium.Iftheentirecontentsarenotneededforasingleprocedure,transferonlytherequiredvolumetoasterilesecondarycontainer.Donotfreeze. QualityControlNHDFandNHLFareculturedwithoutantimicrobialagentsandassayedtoensuretheabsenceofmicrobialcontaminationaftercryopreservation. SubcultureReagentStorage1.Subculturereagentsarestoredat-20·CuntilshippedfromBioWhittaker"sDistributionCenters. 2.Subculturereagentsmaythawduringtransport.Theymayberefrozenonce. 3.Subculturereagentscanbestoredat-20·Cforuptooneyearafterthawingonceandrefreezing. 4.TokeepTrypsin/EDTAfreshandactiveafterthawing,youshouldaliquotitintofive20mlsterilecentrifugetubesandrefreezeat-20·C.Trypsin/EDTAmaybestoredfrozenuptooneyear. 5.WerecommendthatHEPES-BSSandtheTrypsinNeutralizingSolution,oncestoredat4·C,beusedwithinonemonth. HandlingPrecautionsNormalhumancellsarefragile,andrequirespecialhandling: SafetyPrecautionsBioWhittakerstressestheimportanceofthefollowingprecautions: Theflowchartonthefollowingpageillustratesthecultureprocess.Itisfollowedbythestep-by-stepinstructions... InstructionforCryopreservedCells BeforeYouBeginPerformthefollowingstepsbeforeyoubeginmediumorcellpreparation: *Maynotbenecessaryforallend-userassays. MediumPreparationPerformthestepsbelowinasterilefield."Sterilefield"isdefinedabove. FortheFGM®andFGM®-2BulletKits®,dothefollowing: NOTE:Ifthereisconcernthatsterilitywascompromisedduringthesupplementationprocess,theentirenewlypreparedgrowthmediummayberefilteredtoassuresterility.Ifyourefilter,useasterile0.2mm,lowproteinbindingfilter.Routinerefiltrationisnotrecommended. SetUpTosetupvesselsforNHDFandNHLFcomingoutofcryopreservation,dothefollowing: 1.Calculatethenumberofvesselstobesetup.RefertoyourCertificateofAnalysisfortheexactnumberofcellsinyourcryovial.RefertoAppendixE,GrowthAreaofCommonPlasticware,forhelpinadjustingthiscalculation. NOTE:Flasksandmultiwellplatesaremosteffectivetosubculturethesecells. Usethefollowingcalculationstodeterminethenumberofvesselstobesetupfortherecommendedseedingdensityof3500cells/cm2forNHDFand2500cells/cm2forNHLF. No.ofcellsavailable/3500cells/cm2=max.surfaceareathatcanbeplated Max.surfaceareathatcanbeplated/Effectivegrowthareaofflask=max.no.offlasksthatcanbesetup 520,000/3500=148cm2 IfyouuseaT-25withaneffectivegrowthareaof25cm2 148cm2/25cm2=5flasks(roundeddowntonearestwholenumberofflasks) AtypicalcryovialcanbeplatedintoatleastfiveT-25flasks.TheadvantageofsettingupfiveT-25flasksfromtheinitialcryovial,asopposedtolargerflasks,isthatitreducestheriskoflosinglargenumbersofcells.Thatis,ifyouexperiencedifficultytrypsinizingthefirstT-25flask,therearemoreT-25flaskstouse. 2.Labeleachflaskwiththepassagenumber,celltype,strainnumber,anddate. Example:Forfirstpassageoutofcryopreservationforlungfibroblastswithstrainnumber5099,thelabelmightappearasfollows: 3·NHLF5099;12/30/96 3.Inasterilefield,carefullyopenthesupplementedbottleofgrowthmedium,andasepticallytransferthemediumtonewculturevesselsbyadding1mlgrowthmediumforevery5cm2surfaceareaoftheflask. Example:5mlgrowthmediumfora25cm2flaskor60mmplate. 4.Placecapsonvesselslooselyifventedcapsarenotbeingused(i.e.,twistcapsuntiltight,thenloosenabout*turn).Allowtheculturevesselstowarmandequilibrateina37·C,5%CO2,humidifiedincubatorforatleast30minutes. ThawingNOTE:Ifmorethanonecryovialistobethawed,thawonecryovialatatimeandkeepothercryovialsinliquidnitrogenuntilreadyforuse. Aftertheflaskshaveequilibratedfor30minutes: SeedingAftercellsarethawed: NOTE:DonotdispensetheentirecontentsofthecryovialintooneT-25flask!! MaintenanceAfterSeedingNormalHumanFibroblastsarenottolerantofrapidtemperaturefluctuationsornutrient-deficientmedium.Feedingthemwithfreshgrowthmediumthathasbeenwarmedwillavertpotentialproblems.(Remembertowarmonlytheamountneeded.)Checkandfeedthecellsontheschedulebelow,evenonweekendsandholidays. 1.Changethegrowthmediumthedayafterseeding(toremoveresidualDMSOandunattachedcells),theneveryotherdaythereafterwhileexaminingthemdaily. NOTE:Achangeofmediumrequiresremovalofthemediumbyaspiratingwithasterilepipetteontheoppositesideoftheflaskfromwherethecellsareattached.Thenwarm,freshmediumisaddeddownthatsameside. 2.Successfullyrecoveredcultureswillexhibitthefollowing: a.Cellswithclearnon-granularcytoplasm. b.Numerousmitoticfiguresafterday2. 3.Feedthecellsalargervolumeofmediumastheybecomemoreconfluent.Usethistableasaguideline: 4.Continuefeedingthecellsuntil70-90%confluence.Ifthecellsareallowedtobecomeover-confluenttheywillsuffercontactinhibitionandwillpopofftheflaskand/orbedifficulttotrypsinize. OverviewofSubculturePreparation SubculturePreparationNOTE:Thefollowinginstructionsarefora25cm2flask.AdjustallvolumesaccordinglyPreparationforothersizeflasks. Preparationforsubculturingthefirstflask: SubculturingSubcultureoneflaskatatime.Allflasksfollowingthefirstflaskwillbesubculturedfollowinganoptimizationofthisprotocol(explainedlaterinthisprocedure),basedoncalculatedcellcount,cellviability,andseedingdensity. Inasterilefield: AssessingYieldandViabilitySeveralfactorscontributetolowcellcountandlowcellviability.Anexampleofyieldviabilityassessmentisprovidedinthechartbelow.Todeterminethereasonforlowyield/viability,followthesesteps: 1.Studythesamplechartbelow.Itisasampleofhighyield,highviability. a.Notethe"soliddot"onthefar,leftsideofthesquare.Itindicateshighyield,oracellcountofmorethan250,000forNHDFandmorethan500,000forNHLF. b.Notethe"soliddot"ontheXaxisorbottomlineofthesquare.Itindicateshighviability,ormorethan50%viability. c.Extendalinefromeachdotasshowninthechart.Thepointwherethelinesintersect(thebold"X")islocatedintheHighYield/HighViabilityquadrant.Thus,thesampleisoptimal. 2.Now,usingtheblankdiagrambelowplotyourcellyieldandcellviability.Followthesesteps: a.Marka(·)ontheYaxistoindicatethetotalcellcountofyourculture. bMarka(·)ontheXaxistoindicatethecalculatedpercentviabilityofyourculture. 3.Ifyourresultfallsintoanyquadrantotherthanthe"HighYield-HighViability"quadrant,refertoAppendixD,ImprovingCellYieldandViability,beforeproceedingtoyournexttrypsinization. 1.Examinethecellsmicroscopically.Atleast60%ofthecellsshouldhaveattachedtothecultureflask. Somecellswillbelooselyadherent,butmostwillhavespreadoutonthecultureflasksurface.Atthisstage,mostcellswillbesingleorinsmallcolonies. 2.Changetheculturemediumtoremoveresidualtrypsinandnon-attachedcells. 3.Incubateforanadditional24hours,andre-examinetheculture. a.Atthisstage,thevesselshouldhaveseveralmitoticfiguresindicatingthatthecellshaveresumedactivegrowth. b.Iffewmitoticfiguresareobserved,contactyourClonetics®TechnicalSpecialistforassistance. 4.Changethemediumagain48hoursaftertheday1feeding,andevery48hoursthereafterwhileexaminingtheculturedaily. 5.Feedwithvolumesasoutlinedinthetableonpage13. 6.Passageagainwhenthecellsare70-90%confluent.(Ifseededattherecommendedseedingdensity,thisshouldtake5-9days.) InstructionsforProliferatingCells CellPreparation:ProliferatingCellsWiththeproliferatingcultureofNHDForNHLFyoureceived,dothefollowing: SubculturingExamineyourculturesmicroscopicallyeveryday. BIBLIOGRAPHY 1)PolymeraseChainReaction(PCR)technologyiscoveredbyU.S.Patents4,683,195,4,683,202,and4,965,188ownedbyHoffmanLa-Roche,Inc. 2)Cytokeratin18&19.CallyourClonetics®TechnicalSpecialistforareferenceonthisprocedure. 3)Wagner,D.D.,Olmsted,J.B.andV.J.Marder.(1982)ImmunolocalizationofVonWillebrandProteininWeibel-PaladeBodiesofHumanEndothelialCells.JournalofCellBiology.,95:355-360. 4)Grizzle,W.E.,andS.S.Polt.(1988)GuidelinestoAvoidPersonnelContaminationByInfectiveAgentsinResearchLaboratoriesThatUseHumanTissues,J.ofTissueCultureMethods,Vol.11,No.4. BioWhittakerInc.Clonetics®Products9245BrownDeerRoadSanDiego,CA92121(800)852-5663 INTERNATIONALTECHNICALSERVICE: BioWhittakerInc.CloneticsProducts8830BiggsFordRd.Walkersville,MD21793-0127301-898-7025FAX:301-845-2924E-mail:techsup@biowhittaker.com BioWhittaker,Inc.Clonetics®Products8830BiggsFordRoadWalkersville,MD21793(800)344-6618 APPENDIXAOVERVIEWOFFIBROBLASTMEDIA 500mlBottles(exceptwhereindicated) 1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml 1mg/mlhFGF(humanrecombinantFibroblastGrowthFactor)(CC-4065)0.5ml5mg/mlInsulin(CC-4021)0.5ml50mg/mlGentamicin,50mg/mlAmphotericin-B(CC-4081)0.5ml10mlFBS(FetalBovineSerum)(CC-4101) 3.513mg/mlL-Cysteine(CC-4069)2ml6.559mg/mlL-Isoleucine(CC-4045)1ml9.01mg/mlMyo-Inositol(CC-4050)1ml9.838mg/mlL-Leucine(CC-4046)2ml2.238mg/mlL-Methionine(CC-4047)1ml2.878mg/mlL-Proline(CC-4048)1ml0.036mg/mlL-Thymidine(CC-4049)1ml 3.513mg/mlL-Cysteine(CC-4069)2ml6.559mg/mlL-Isoleucine(CC-4045)1ml9.01mg/mlMyo-Inositol(CC-4050)1ml9.838mg/mlL-Leucine(CC-4046)2ml2.238mg/mlL-Methionine(CC-4047)1ml2.878mg/mlL-Proline(CC-4048)1ml0.036mg/mlL-Thymidine(CC-4049)1ml NOTE:AllClonetics®Mediacanbecustomformulatedtomeetyourresearchneeds.ContactyourTechnicalSpecialistformoreinformation. APPENDIXBCELLCOUNTINGUSINGAHEMACYTOMETER ProperuseofahemacytometeriscriticalforobtaininganaccuratecountofcellsandisaprocedureusedbyBioWhittakertodeterminethesuspensioncountsforClonetics®cellstrains.Ahemacytometerconsistsofathic,kenedglassslideintowhichasmallchamberhasbeencuttoallowfortheintroductionofcellstobecounted.Thefloorofthechamberisdivided(etched)intoninesections;usuallyonlythefourcornersectionsareusedincellcounting(SeeFigure1below).Withacoverslipinplace,eachsquareofthehemacytometerrepresentsatotalvolumeof0.1mm3or10-4cm3.Since1cm3isapproximatelyequivalentto1ml,thecellconcentrationperml(andthetotalnumberofcells)canbedetermined. 1.Prepareacellsuspensionasinstructedinstep13onpage16. 2.Prepareahemacytometerforuse. a.Carefullycleanallsurfacesofthehemacytometerandcoverslip. b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue. c.Centerthecoversliponthehemacytometer. 3.Pipetapproximately9microliters(thisvolumewillvaryslightingwiththebrandofhemacytometer)ofthecellsuspensionintooneofthetwocountingchambers. a.Useacleanpipettip. b.Besurethatthesuspensionisthoroughly,butgently,mixedbeforedrawingthesamples. c.FillthechambersslowlyandsteADIly. d.Avoidinjectingbubblesintothechambers. e.Donotoverfillorunderfillthechambers. 4.CounttheCells. a.Allowthecellsuspensiontosettleforatleast10seconds. b.Countallofthecellsineachofthefour1mm3cornersquareslabeledAthruDinFigure1onthenextpage. 1)DOcountthecellstouchingthetoporleftborders. 2)DONOTcountthecellstouchingthebottomorrightborders. 5.DeterminetheCellCount. a.Calculatethetotalcellscountedinthefourcornersquares. 1)Ifthetotalcellcountislessthan100,orifmorethan10%ofthecellscountedappeartobeclustered,carefullyre-mixtheoriginalcellsuspensionandrepeatsteps2through4(above). 2)Ifthetotalcellcountisgreaterthan400,dilutethesuspensionsothecountwillbe100-400cells.ThenrepeatSteps2-4(above). NOTE:Ifsatisfactoryresultsarenotachieved,contactyourClonetics®TechnicalSpecialistbytelephoning800-852-5663. b.Calculatethecellcountusingtheequation:cells/ml=(n)x104, where:n=theaveragecellcountpersquareofthefourcornersquarescounted. Example:Ifthecalculatedaverage(n)ofcellsinthefour1mmcornersquaresofthehemacytometeris30: cells/ml=(n)x104(or)cells/ml=30x10,000=300,000cells/ml. c.Determinethetotalnumberofcellsinthetotalsuspensionvolume. 1)Determinethetotalvolumeofthecellsuspension. 2)Multiplythevolumeofthecellsuspensionbythe"cells/ml"valuecalculatedabove. Example:Iftheinitialsuspensionvolumeis2ml: cells/mlxtotalvolume=300,000cells/mlx2ml=600,000cells. APPENDIXCASSESSMENTOFCELLVIABILITYWITHTRYPANBLUE Trypanblueisadyethatenableseasyidentificationofdeadcells.Deadcellstakeupthedyeandappearbluewithunevencellmembranes.Bycontrast,livingcellsrepelthedyeandappearrefractileandcolorless. 1.Preparethehemacytometerforuse. a.Carefullycleanallsurfacesofthehemacytometerandcoverslip. b.Takecaretoensurethatallsurfacesarecompletelydryusingnon-lintingtissue. c.Centerthecoversliponthehemacytometer. 2.Transfer50mlof0.4%TrypanBlueintoacleantube. 3.Add50mlofthepreparedcellsuspensionintothetubecontainingthestain. 4.Mixthesolutionthoroughly,butgently.Takecaretoavoidmakingexcessivebubbles. 5.Allowthemixturetositfor2-3minutesaftermixing.(Donotletthecellssitinthedyeformorethanfiveminutesbecauseboththelivinganddeadcellswillbegintotake-upthedyeafterfiveminutes.) 6.Pipetapproximately9microlitersoftheTrypanBlue/cellsuspensionmixture(thisvolumewillvarywithbrandofhemacytometer)intooneofthetwocountingchambers. a.Useacleanpipettip. b.Besurethatthesuspensionismixedthoroughlybutgentlybeforedrawingthesamples. c.Fillthechambersslowlyandsteadily. d.Avoidinjectingbubblesintothechambers. e.Donotoverfillorunderfillthechambers. 7.DetermineCellViability. a.Allowthesuspensiontosettleinthechambersforatleast10seconds. b.Countallofthestainedcellsineachofthefourcornersquaresofthehemacytometer. c.Separatelycountalloftheunstainedcellsinthesamesquares. d.Calculatethecellviabilityusingtheequation: %CellViability=numberofunstained(living)cells/Totalcellscounted(stained+unstained)x100% Example:Ifatotalof300cells(stained+unstained)arecountedand200areidentifiedaslivingcells(unstained),thentheviabilityiscalculatedas: %Cellviability=200/300x100%=67% IMPROVINGCELLYIELDANDVIABILITY Background Severalfactors,oracombinationoffactors,contributetolowcellcountandlowcellviability.Ifcellyieldorviabilityisunsatisfactory,usethefollowinginformationtoincreasethesuccessrateoffuturecultures. ImprovingCellYield Ifyourcellyieldislow(lessthan50%),determinethecause(s)andpossiblesolution(s)usingthetablebelow.Thensubcultureonemoreflaskapplyingtheappropriatesolution(s). Ifyourcellviabilityislow(lessthan50%),determinethepossiblecause(s)andsolution(s)usingthetablebelow.Thensubcultureonemoreflaskapplyingtheappropriatesolution(s). Onceyouhavedeterminedhowtoachievehighyieldandhighviability,subculturetheremainingflasks. APPENDIXEGROWTHAREAOFCOMMONPLASTICWARE APPENDIXFSeedingIntoMulti-WellPlates Overview Acultureflaskofnormalhumancellsisharvestedbytrypsinizationandsubsequenttrypsininhibitortreatment.Thecellsarecentrifuged,resuspendedingrowthmediumandcounted.ThedesirednumberofcellsisthenaddedtowellsofsterileMulti-welltissuecultureplates.Theplatesareincubatedina37oC,5%CO2humidifiedincubatorforonetothreedaystoallowforcelladherenceandgrowth.Seedingdensitieswillvarysomewhatwithyourexperimentalrequirements.WerecommendadensityforDermalFibroblastsandLungFibroblastsof10,000cells/cm2forallmultiwellplates. RequiredMaterials: Procedure NOTE:BeforeusingtheMulti-wellplatecultureinabioassay,examinethemmicroscopicallyforthepresenceofmitoticfiguresasaconfirmationthatthecellshaveresumedactivegrowth.(Doesnotapplyforallend-userassays.) FaxOrderForm (Pleaseprintortype) DateofFax: BillingAddress: Attn.: PurchaseOrderNo.: Company: FaxNumber: VoiceNumber: ShippingAddress: Attn.: DateRequired: (1)PolymeraseChainReaction(PCR)technologyiscoveredbyU.S.Patents4,683,195,4,683,202,and4,965,188ownedbyHoffmanLa-Roche,Inc.CC-2511 NHDF-Ad 3500,000cells/cryovial CC-2509 NHDF-Neo 3500,000cells/cryovial CC-2512 NHLF 3500,000cells/cryovial CC-2611 NHDF-AdT-25flask »Yield:500,000cells CC-0252 NHDF-AdT-75flask »Yield:1,500,000cells CC-2609 NHDF-NeoT-25flask »Yield:500,000cells CC-0210 NHDF-NeoT-75flask »Yield:1,500,000cells CC-2612 NHLFT-25Flask »Yield:1,000,000cells CC-0282 NHLFT-75Flask »Yield:3,000,000cells ProliferatingCellsinPreseeded®96-wellPlates
CC-0160 NHDF-Ad 96wells CC-0116 NHDF-Neo 96wells CC-0164 NHLF 96wells ProductName NormalHumanCellType CryopreservedAndProliferatingCellsProductNumbers RecommendedMedia NHDF AdultDermalFibroblasts CryopreservedCC-2511 FGM®-2BulletKit® NHDF NeonatalDermalFibroblasts CryopreservedCC-2509 FGM-2®BulletKit® NHLF LungFibroblasts CryopreservedCC-2512 FGM®-2BulletKit® GeneralInformation
FGM®BulletKit®(CC-3130)andFGM®-2BulletKit®(CC-3132) HowPrepared StorageRequirements ShelfLife AllFGM®BulletKit®andFGM®-2BulletKit®componentshavebeenhumancellculture-tested.Allsolutionsaresterile-filteredbypassagethrougha0.2mmfilter.Basalmediumisstoredat2·to8·C,andgrowthfactorsarestoredat-20·Cuntilshipment. Ifthaweduponarrival,growthfactorscanbestoredat2·to8·CandaddedtoFBM®within72hoursofreceipt. FGM®BulletKit®andFGM®-2BulletKit®shelflifearelimitedbytheshelflifeoftheFBM®,whichis1yearfromthedateofmanufacture.Whengrowthfactorsareaddedatanytimewithinthistimeperiod,werecommendusewithin1month,butbeforethebasalmediumexpiration. CellType VonWillebrandFactor SmoothMuscleAlphaActin Cytokeratins18&19 NHLF Negative Negative Negative SafetyPrecautions
Asaprecautionagainstcontamination,followallproceduresforhandlingproductsofhumanoriginoutlinedin"GuidelinestoAvoidPersonnelContaminationByInfectiveAgentsinResearchLaboratoriesThatUseHumanTissues,"fromtheJ.ofTissueCultureMethods.4(SeeBibliography,page21.) Alwayswearglovesandsafetyglasseswhenworkingwithallmaterials.Exercisecautionwhenworkingwithcryopreservedcells;rapidtemperaturechangesmaycausesplatteringofliquidnitrogen. Washhandsthoroughlyafterperformingallprocedures. Nevermouthpipet. Donotsmoke,eatordrinkinareaswherereagentsorcellsarehandled. Productsofhumanoriginarepotentiallybiohazardous.AlthougheachcellstraintestsnegativeforHIV-1,hepatitisBandHepatitisC,properprecautionsmustbetakentoavoidinadvertentexposure. Prepareasterilefield. AsterilefieldconsistsofaClassIIbiologicalsafetycabinetwithafrontaccessopeningandfilteredlaminarairflow,orothersuchequivalentdevice. Determinetheamountofmediumrequired. ReviewtheGrowthAreaofCommonPlasticwareChart(AppendixE)todeterminetheamountofmediumtobeused. Collectsterileinstrumentsandvessels. Collectothersupplies. Planandprepareforinitialsetup. BaseyoursetuponthenumberofcellsindicatedontheaccompanyingCertificateofAnalysis.(SeeAppendicesBandC.) Checkthecalibrationonhumidifiedincubator. Incubatorshouldbea5%CO2/95%air,humidifiedincubator,setto37·C. Example:Acryovialwith520,000cells
Under25%confluent... 1mlper5cm2 From25-45%confluent... 1.5mlper5cm2 Exceeding45%confluence... 2mlper5cm2 ORDERS:
CC-3131 FBM® FibroblastBasalMedium(nogrowthfactors) CC-3130 FGM®BulletKit® Kitwhichcontainsa500mlbottleofFBM®,(CC-3131)andFGM®SingleQuots®(CC-4134)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-usealiquots(amountsindicateconcentrationofeachSingleQuot®) CC-3132 FGM®-2BulletKit® Kitwhichcontainsa500mlbottleofFBM®,(CC-3131)andFGM®-2SingleQuots®(CC-4126)whichcontainsallofthesupplementslistedbelow,convenientlypackagedassingle-usealiquots(amountsindicateconcentrationofeachSingleQuot®) CC-3134 FGLM™Custom FGM®LabelingMediumBulletKit®(500ml)thatconsistsofthefollowing: CC-3133 FBLM™FibroblastBasalLabelingMedium,withoutthefollowingnutrients:Myo-Inositol,Thymidine,Proline,Isoleucine,Leucine,Methionine,andCysteine. CC-4153 FGLM™SingleQuot®Kit,FGM®labelingSingleQuots®consistingofthefollowing: CC-4134 FGM®SingleQuots®(SeeCC-3130) CC-3135 FGLM™-2Custom FGM®-2LabelingMediumBulletKit®(500ml)thatconsistsofthefollowing: CC-3133 FBLM™FibroblastBasalLabelingMedium,withoutthefollowingnutrients:Myo-Inositol,Thymidine,Proline,Isoleucine,Leucine,Methionine,andCysteine. CC-4153 FGLM™SingleQuot®Kit,FGM®labelingSingleQuots®consistingofthefollowing: CC-4126 FGM®-2SingleQuots®(SeeCC-3132) Majorityofcellsdidnotdetach. 95%ofthecellsdetachedbuttheyieldwaslow. Culturewasunderconfluentattrypsinization. Besuretotrypsinizeat70-90%confluencewithatleast5mitoticfiguresperfieldofview. Trypsin/EDTAdamagedthecells. Culturevesselwastooconfluent. Culturewastooconfluentattrypsinization. Besuretotrypsinizeat70-90%confluencewithaboutfivemitoticfiguresperfieldofview. Cellgrowthslowedbefore90%confluenceandcellslookdullandnon-refractile. Themostprobablecauseisfailuretoincreasethevolumeofmediumusedasthecellconfluencyincreased.Thecellsbecomemildlystarvedandarenotabletorecoveraftertrypsinization. Changemediumandincreasevolumeasrecommended.Pleaseobserveallguidelines. T-25 25cm2 87,500 500,000 62,500 1,000,000 T-75 75cm2 262,500 1,500,000 187,500 3,000,000 T-150 150cm2 525,000 3,000,000 375,000 6,000,000 35mm 9.6cm2 33,600 192,000 24,000 384,000 60mm 28.0cm2 98,000 560,000 70,000 1,120,000 100mm 78.5cm2 274,750 1,570,000 196,250 3,140,000 150mm 176.6cm2 618,100 3,532,000 441,500 7,064,000 6well 9.60cm2 96,000 192,000 96,000 384,000 12well 3.80cm2 38,000 76,000 38,000 152,000 24well 2.00cm2 20,000 40,000 20,000 80,000 48well .75cm2 7,500 15,000 7,500 30,000 96well .32cm2 3,200 6,400 3,200 12,800 To:BioWhittaker,Inc.Clonetics®ProductsCustomerService Fax:(301)845-1008
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