I.Purpose:

Amnioticfluidmaybeusedforprenataldiagnosisofaneuploidyorotherstructuralabnormalities.

II.CultureProcedure:

A.Aseptictechniquemustbeusedwhensettinguptheculturesunderthelaminarflowhood.

B.CulturesaregrowninamixtureofCHANGandF-10.Themediamustbefresh(lessthan4daysold),prewarmed,andatpH7.0-7.5.Allculturesareincubatedinawet,5%CO2incubatorat37C.

C.Specimensareusuallyreceivedintwo15mlconicalcentrifugetubes.Ifthesampleisreceiveddifferently,transfertotwotubes.Centrifugethetubesfor6-8minutesat150xg(900rpm).Leaving0.5ml,transfersupernatanttoa15mlcentrifugetube(thiswillbefrozenforback-upAFPstudies).Ifspecimenisneededforprenataltests,save2mlofsupernatantinavial(liketheonesusedfortissuetransport).Parafilmthevial,placeinspecimenbagandputinfridge.PleasenotifyKate,Sue,orDr.SekhonofsampletogotoSLH.

D.Label30mmpetridishesw/coverslipswithlab#,patientname,AF,dateset,andculture(AorB).Routinely,set4coverslipspertube.Forsmallpellets,set3coverslipspertube.Ifonlyonetubewassent,set6coverslipsfromthatonetube.

E.ResUSPendpelletinmedia(4coverslips,add1.5ml;3coverslips,add1.0mlofmedia).Add0.5mltoeachcoverslip,beingcarefultokeepitonthecoverslip.Dothesameforeachtubeofamnioticfluid.Set-updayisday0.Place"A"coverslipsintoincubator#3,and"B"coverslipsintoincubator#2.

F.Onday2floodthecoverslipswith1.5mlofmedia.AandBculturesshouldbefedwithappropriatebottlesofmedia,eachlabelledAorB.Onday4feedwith0.5mlofmedia.

G.Startingonday4,checkforgrowth.Ideally,thiswillbeasindividualcoloniesdispersedoverthecoverslips.Onday5or6,dependingonthenumberandsizeofcolonies,mediashouldbechangedandreset.Mediashouldbechangedwhencoverslipscanbeharvestedthefollowingday.OnecoverslipshouldbereservedfromharvestforpossIBLesubculturing.(If,onday6,coverslipsarenotreadyforafullmediachange,carefullyaspirateoff1mlofmediapercoverslip,discard,andrefeedwith1mlfreshmediausingtheappropriateAorBmediabottle.Followthesecoverslipsuntilreadyforafullmediachangeandreset.)

H.ChangethemediabyusingasterilePipettetotransferthesuspensionintoasterile15mlconicaltube.(All"A"coverslipsinonetube,andall"B"coverslipsintoasecondtube.)Rinsethecoverslipsusing2mlfreshmedia(usingtheappropriateAorBmedia).Therinsemediashouldalsobesavedinthe15mltube.Rinsetwiceifnecessary.

Add2mlmediatoeachdishandplaceintheincubator.

RESETPROTOCOL

1.Centrifugefor6-8minutesat150xg(900rpm).Discardthesupernatant.Foreachtube,resuspendthepelletusing1.0mloffreshmediaandplateonto2labeledcoverslips.(ifthepelletislarge,resuspendin1.5mlofmediaandplateonto3coverslips).Coverslipsshouldbelabeledwithlabnumber,AF,cultureAorB,dateand"reset".

2.Onday2,floodthecoverslipswith1.5mlmediaagainusingtheappropriateAorBmedia.

3.Onday3,feedthecoverslipswith0.5mlmedia.Alsocheckthemforpossiblemediachange.(Resetsgrowfasterthanprimaries.)

4.Onday4,checkforamediachangeandharvest..Discardthismedia.Noresetisrequired.

I.CoverslipSubcultures-ifnecessary,coverslipculturesmaybesplit(subcultured)ontomorecoverslipstoimprovegrowth.Thisisdonebythefollowingprocedure:

-Removeanddiscardmediafromcoverslips

-Add0.2mltrypsin,letsit20sec(rockinggently)

-Removeanddiscardtrypsin

-Add0.2mltrypsin,incubate5min

-Gentlyknockoffcells

-Using2mlmedia(Chang/F-10),washalldishesandtransfertoasterile15mlconicalcentrifugetube.

-repeatwithanadditional2mlmedia

-Centrifugetube

-Resuspendin0.5mlmediapercoversliptobeset.

-Floodcoverslipsatendofdaywith1.5mlofmediaandharvestonetotwodaysfollowingsetup

III.HarvestProcedure:

A.Startingonday5checkthecoverslipstoseeiftheyarereadyforharvest.Thereshouldbemultiplecolonieswithrounded,dividingcellsoneachcoverslip.IfthecoloniesareallowedtogrowtoolargetheymaygrowtoodenseorintooneanotherandtherewillbetoomuchcytoplasmforgoodspreADIngandbandingofthechromosomes.Ifthecoloniesaretoosmall,insufficientmetaphasesmaybefound.

B.Add20µlEthidiumBromideworkingsolutiontoeachdish,incubatefor40minutes.Add40µlofcolcemidworkingsolution,incubateforanother20minutes.

C.Tenminutesbeforeendofincubation,startupTECANharvestersothatitwillbeready.

D.HarvestusingTECAN.

E.Dryingconditionsareveryimportantfortheproperspreadingofmetaphases.UsingtheTECANdryprogram,removefixativefromthedish.Usingtheaspirator,removealmostallthefix,goingaroundtheedgeofthecoverslip.AllowtodryinthePercivalScientificdryingchambersetat35.0%relativehumidity,28.0°C,fanspeed72-75%.Ifthecoverslipdriestoorapidly,allthecellswillbetrappedinthemembranes.Ifitdriestooslowly,thechromosomeswillfloatawayfromthemetaphase.

F.Removethecoverslipfromthepetridish,keepingitright-sideup.Ithelpstoholdthedishwithyourthumbandmiddlefingerandbendupthebottomofthedishwithyourindexfingersothatthecoverslipisliftedup.Mountthecoversliponalabeledmicroscopeslideusingadropofmountingmedia.Placeonlyonecoverslipperslide,properlylabeledwithpatientnumber,AorBculture,AF,and"reset"ifnecessary.

G.Allowtheslidestodryforatleast30minutesatroomtemperature.Bakeat90°Cfor30-60minutes.SlidesarenowreadyforG,Q,orCbanding.Metaphasesareresistanttotrypsinizationandalsotendtorequiremorestainthanothertypesofspecimens.

V.Solutions:

Colcemidworkingsolution:10mcg/mlColcemidinHank"sBalancedSaltSolution,storeat4C.

CompleteCHANG/F10media:45mlChangbasalmediaB,42mlNutrientMixF-10,5mlChangsupplementA,8mlFetalcalfserum,1mlPenicillin/Streptomycin,solution,1.1mlL-Glutaminesolution,storeat4C.

EthidiumBromideWorkingSolution:2mg/mlEthidiumBromideinRPMI-1640,storedindark.

Fixative:30mlMethanoland10mlGlacialAceticAcid,preparedfresh.

Hypotonicsolution:0.8%NaCitrate/0.075MKCl:1.6gNaCitrate,1.68gPotassiumChloridedissolvedin500mldH2O.

1XTrypsinEDTAsolution:10mlstocktrypsinsolution(10Xtrypsininsaline),0.1gEDTA(disodiumsalt),dissolvedin490mlHank"sBalancedSaltSolution.Sterilefiltered,50mlaliquots.Storefrozen.

VI.Reagents::

Changmedia:Irvinecat#T100-018,liquidandfrozensupplement,100mlbottle,storesupplementfrozen,basalmediaat4C.

Colcemid:Gibcocat#15210-040.10mcg/mlinHank"sBalancedSaltSolution,10mlbottle,storeat4C.

EthidiumBromide:SIGMAcat#E-8751.2,7-diamino-10-ethyl-9-phenyl-phenanthridiniumbromide,250mgbottle.

Fetalcalfserum:HYCLONE/STERILESYSTEMSINC.cat#A-1111-D.definedfetalbovineserum,100mlbottle,storefrozen.

GlacialAceticAcid:FISHERcat#A38-500.Aceticacid,GlacialACS(Aldehydefree),500mlbottle.

L-Glutamine:GIBCOcat#320-5030.L-Glutaminesolution100X(200mM),20mlbottle,storefrozen.

Methanol:FISHERcat#A412-500.MethylalcoholanhydrousAR(ACS)(Absolute)Acetonefree,500mlbottle.

NutrientMixF-10:GIBCOcat#81200-040.NutrientMixtureF-10(HAM)powderedform,preparedtoinstructions,10x1literpackage.

Penicillin/Streptomycin:GIBCOcat#600-5140.Penicillin/Streptomycinsolution,10,000units/ml/10,000mcg/ml,20mlbottle,storefrozen.

PotassiumChloride:COLUMBUSCHEMICALINDUSTRIES,INC.ACSgranular,500glots.

SodiumCitrate:UW-Stores,cat#2588.Crystal,ACS,Na3C6H507.2H20,500gbottle

TrypsinStockSolution(1X):GIBCOcat#25300-013.Trypsin2.5%(10X)insaline.Storefrozen

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