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