Transfer of Eukaryote Suspension Cultures
来自 : 蚂蚁淘
TransferofEukaryoteSUSPensionCulturesLEVELII
Materials
FibroblastsuspensioncultureTissueculturelaminarflowhoodMediaappropriatetoculturelineusedDisposablePipettes(10mland1.0ml)DisposablecultureflasksProcedure
- Obtainacultureofmousefibroblastcellsinsuspensionculture.Thiswillbeasimpleculturewithminimalrequirements,andoneselectedforexcellentgrowthcharacteristics.Thetransferprocedurewillbesimilartothatforprokaryotes,withafewmajorchanges.First,alltransferswillbedoneinatissueculturehoodinordertomaximizeasepsis.Secondly,thecellswillbetransferredwithsiliconizedpipettesratherthanwireloops.Thesiliconepreventsadheranceofthecellstotheglasswallofthepipette.
Atissueculturehoodisadevicethathasairmovinginlayersandunderpositivepressure.Sincetheairisfiltered,itcontainsminimalnumbersofbacteriaorfungalspores,andsinceitisunderapositivepressure,thoseparticlesthatarepresentareblownoutofthehood.Thelayeringpreventsairborneorganismsfromsettlingontheworksurfaces.
- Arrangethematerialsinfrontofyou,easilyaccessIBLethroughtheopeningofthetissueculturehood.Ensurethatanyalcoholsandwrappingpaperarekeptclearofthebunsenburner.Pre-sterilizethehoodbeforeuse,andusedisposablesterilegloves.
- Loosenthecapofatissuecultureflaskandthecapofastockbottleoftissueculturemedia.6
Insertthetipofasterilepipetteintothestockbottleandremove10mlofmedia.Transferthemediatothetissuecultureflask.
- Openthetopofthesuspensioncultureanduseasterile1.0mltransferpipettetoremovea1.0mlsampleoftheculture.Transferittothefreshmediainthecultureflask.Secureallcapsthathavebeenloosened.
- Placethenewculturesinanincubatorat37°C.
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