Organotypicraftcultureofprimaryhumankeratinocytes(PHKs) Notes Use106keratinocytesperraft. TypicalProtocol TrypsinizethePHKsofftheflaskandresUSPendin(say)2mlraftmedium. Seeseparateprotocolforraftmedium RemovethemediumfromthecollagenplugsandoverlaywiththenewmediumcontainingPHKs.Incubatefor1-2daystoallowthePHKstosettleandbecomeconfluent.Autoclavethefollowing; SpatulasforscoopingoutthecollagenplugPBSStainlesssteelmeshgridinaglasspetri-dish.(Thoroughlyrinsetheglasspetri-dishtoremovedetergent.Makethefour"legs"forthemeshgridbyplacingitflatontoamicrofugetuberackandpressingatubeintoanedge,deformingitdownwards.Thisdownwarddeformedbitisaleg.) WhenthePHKsareconfluent:-Separatetheedgeofthecollagenplugfromthewallofthewellbygoingrounditwithasterilescalpel.Allowthecollagenplugtocontractawayfromthewallsforafewhours. Thisislikecuttingroundacakeinacaketin.ThePHK"membrane"maycontractfromtheedgealsoanddetachalittlefromthecollagen Removemedia,scoopoutpluganddepositontothemeshinthepetri-dish,keepingeverythingsterile. Optional-rinseinPBSbeforeplacingontomeshThistransferisthetrickiestpartofthewholeprocedure.ThereisnoeasywaytodoitalthoughIfoundthatthecollagengelisstrongerthanitlooks.Ifyougetitintact,rightsideup(ie.PHKsup),andlookingroundishonthemesh,withouthavingscrapedoffallthePHKs,thenyou"vedonewell.Usuallyitendsuponthefloor.SometimesthePHKsallcomeawaylikeamembrane;trytospreaditbackontothecollagen.Keepsterile Feedtheraftbyadding12-15mlofraftmediumtothepetri-dish. Theideaistowettheundersideofthemeshbutnottolettheliquidcomeontotheuppersurface.IfthetopoftheraftbecomeswetitwilldestroythegrADIentandspoiltheexperiment.Watchoutforairbubblesloiteringundertheraft. RefeedeverytwodaysPeopleseemtoleavethemfor8-14daystogrow/differentiateAddinBrdU8-12hoursbeforeharvesting(seeseparateBrdUprotocol) Suppliers Stainlesssteelwiremesh31/16inch40mesh.010(=wiresize)304SScircle,item#105493100forabout$25from;USFilter-Johnson/NiagaraScreen(acivilengineering,waterfiltertypecompany)NiagaraMeshSalesFax0017139391337Tel0017139391830
