AsepticTechnique

·AsepticTechnique(ContributedbyNanciDonacki)AseptictechniquesusedbyCellCulturespecialistsinhandlingproductsfromand/ormammaliancells.

CultureVessels

·Recommendedworkingmedium,trypsinvolumeandcellinoculationdensityUsefuldataforcellsubculturewithdifferentculturevessels.

·UsefulNumbersforCellCulture(LTI)Referencetableofusr/localiousculturevesselsandtheirarea,plantingcellnumber,cellyieldandmore

• SurfaceAreasofCellCultureVessels(LTI)

·PlasticDishesforGrowingCells(SeftonLab)

·WorkingVolumesforTissue-CultureVessels(BectonDickinson)Acomprehensivechartfordeterminingappropriatevolumesofmedium,washbuffersandtrypsinforspecifictissueculturevesselconfigurations)

CellCounting

• UseofaHemacytometer(P.J.HansenLab)Verydetailedguideoncountingcellsusingahemacytometer

·HemacytometerReferenceGuide(BectonDickinson)Illustratedinstructionforusingahemacytometertocountcells

·EukaryoteGrowthDynamics(WilliamH.Heidcamp)Fordeterminingcelldoublingtime.

·CellCountsUsingaHemocytometer(Donis-Kellerlab)Thepurposeofthisprocedureistodeterminethecelldensityoftheculture.Cellculturesalwayshavesomedeadcells;theviableandnon-viablecellscanbedistinguishedwiththeuseoftrypanbluedyeandahemacytometer.Livingcellswillnottakeupthedyewhiledeadcellsdo.

·CellCounting(SeftonLab)

·ViableCellCountsUsingTrypanBlue(LTI)ViableCellCountsUsingTrypanBlueViableCellCountsUsingTrypanBlueTrypanblueisavitaldye.Thereactivityoftrypanblueisbasedonthefactthatthechromoporeisnegativelychargedandd

·ViABIlityCellCount(WilliamH.Heidcamp)

PrimaryCulture

·EstablishmentofaPrimaryCulture(WilliamH.Heidcamp)

·CultureofEndometrialExplants(P.J.HansenLab)Thismethodisusedtostudysecretionofproteinsandprostaglandinsbyendometriumfromthecow,ewe,mare,bitchandotherspecies.Thetechniqueisalsousefulforcultureofperi-implantationconceptusesandplacentaltissuesformetaboliclabellingstudiesandtoobtainconceptussecretoryproteinsforBIOLOGicalstudies.

·FractionationofCellTypes(LTI)SeparationofbloodortumorcellsintodifferentcelltypesondensitygrADIentsisdonefrequentlyforestablishmentofprimarycultures.InacellsUSPensioncontainingamixedpopulationofcells,cellscanbeseparatedonthebasisofbothsizeanddensity.

·DissociationofcellsfromPrimaryTissue(LTI)Severalmethodsareintroduced.

MaintenanceofCellLine

·MaintenanceofCellCultureDetailedprocedureforcultureandsubculturecellinflaskandplates

·AdaptationofCellstoSerumFreeMedium(LTI)AdaptationofCellstoSerumFreeMediumAdaptationofCellstoSerumFreeMediumSEQUENTIALADAPTATIONSubculturethecellsgrowinginserum-supplementedmediumintoa1:1(v/v)mixtureofSFMand...

·LymphocyteTransformation(Donis-Kellerlab)Lymphocytesaretransformedtoestablishcelllines.Mononuclearcells(lymphocytes)fromanticoagulatedvenousbloodareisolatedbylayeringontohistopaque.Duringcentrifugation,erythrocytesandgranulocytesareaggregatedbyficollandrapidlysettletothebottomofthetube;lymphocytesandothermononuclearcellsremainattheplasma-histopaqueinterface.ErythrocytecontaminationisneglIBLe.Mostextraneousplateletsareremovedbylowspeedcentrifugationduringthewashingsteps.

·PreparationofLymphoblastoidCellLinesforLong-TermStorage(Donis-Kellerlab)Tostorecelllinesinaformthatwillinsurerecoverywithhighviability.Acultureinlogarithmicphaseofgrowthwithatotalvolumeof80-100ml/T-75flaskshouldyieldenoughcellstofreeze10ampules(1.0ml/ampule).Cellsshouldhaveacountof4X106cells/ampuleto9X106cells/ampule.Toohighortoolowacellcountlowersrecoveryviability.CellarefrozeninRPMI-1640with15%FetalBovineSerum+10%DMSO.CulturesarefrozenslowlyusingaModel700Controllerfreezingchamber.Thisprecisionelectronicdeviceautomaticallycontrolstheinjectionofliquidnitrogenintothefreezingchambertoprovidea1degreesC/minutefreezingratefrom+4degreesCto-45degreesC(withautomaticheatoffusioncompensation),thena10degreesCperminutefreezingrateto-90degreesC.Frozenampulesshouldbestoredinliquidnitrogenforlongtermstorageorina-135degreesCCryopreservationSystem.Note:Cryotubesshouldbelabeledwithcelllinenumberanddatepriortobeginningthisprocedure.

·MaintainingLymphoblastoidCellLines(Donis-Kellerlab)TogrowlymphoblastoidcellsforpermanentstorageandforDNAextraction.

·LymphoblastoidCellLinesfromFrozenWholeBlood(Donis-Kellerlab)BloodSamplescanbestoredfrozenasabackupincaseanLCLisneededatalaterdate.

·CellSuspensionCulture(Gimila"sLab)Tobaccocellculturemethodincludingmediapreparation

·TransferringCHOCellsFromMonolayertoSuspensionCulture(LTI)

·TransferofEukaryoteSuspensionCultures(WilliamH.Heidcamp)

·CultureofBENDCells(BovineEndometrialCells)(PeterJ.Hansen)

·Cultureofendometrialexplants(PeterJ.Hansen)

Hybridoma

·CloningbyLimitingDilution(ContributedbyNanciDonacki)

Cellharvesting

·Non-enzymaticMethodsforCellHarvesting(NUNC)

·PreparationofLymphocyteCellPelletforStorage(Donis-Kellerlab)Followingpropagationto1X108cells,lymphoblastoidcellsareconvenientlystoredat-80degreesCtopreservethehighmolecularweightDNAinthecellsuntiltheDNAispurified.Thisproceduredescribesthestepsrequiredtoharvestandfreezethecellsforlongtermstorage.

·TrypsinizingCells(SeftonLab)

·DissociationofCellsfromCultureVessels(LTI)DissociationofCellsfromCultureVesselsDissociationofCellsfromCultureVesselsThefollowingisageneralproceduretorapidlyremoveusr/localiouscelllinesfromthesubstratumwhilemaintaining...

·DissociationofCellsfromPrimaryTissue(LTI)DissociationofcellsfromPrimaryTissueDissociationofcellsfromPrimaryTissueTRYPSINAfterdissectingoffunusabletissue,mincetheremainingtissueinto3to4mmpieceswithasterile...

• TrypsinizationofAdherentCells(SupryaJayadev)

SpcialCellandTissureCulture

• Cultureofendometrialexplants(P.JHansenLab)
• CultureofBENDcells(abovineendometrialcellline)(P.JHansenLab)
• TissueCultureofPtK₁Cells(SalmonLab,UniversityofNorthCarolinaatChapelHill,DepartmentofBiology)

AntibioticsandContamination

·DecontaminationofCultureswithAntibiotics(LTI)DecontaminationofCultureswithAntibioticsDecontaminationofCultureswithAntibioticsWhenanirreplaceableculturebecomescontaminated,researchersmayattempttoeliminateorcontrolthe...

·UseofAntibioticsandAntimycotics(LTI)providesareferencetableofcommonlyusedantibioticswiththeirconcentrationinmedia,antibioticspectrumandstability

·RemovalofYeastContaminationfromLymphoblastCultures(Donis-Kellerlab)Thismethodisadvantageousforsavingtheoccasionalculturesthatbecomecontaminated.Yeastcontaminatedcultureswillappearcloudywhenslightlyshakenandlymphocyteswillnotclustertogetherasmuchasnormal.Ifculturesaresuspect,adropofculturecanbestreakedonaYPDmediaplatetocheckforgrowthofyeastcolonies,ora5mlsamplecanbetakentoBarnesDiagnosticCenterforidentificationofyeaststrain.

Others

·TobaccoCellSuspensionCultureMethods(GimilaLab)

·Mini-Chamberforregulatinggaseousenvironmentduringculture(P.J.HansenLab)

·LoggingInSpecimensandRecordKeeping(Donis-Kellerlab)Tokeepawrittenandcomputerizedrecordofallcelllines,thedateswhencelllineswerereceivedandfrozen,freezerlocations,andanyotherimportantinformationsuchasdatesofbirth,sex,etc.

• InfectionCellwithRetroviruses(SeftonLab)

• ChecklistforMediaReplacement(BectonDickinson)Thingstorememberandpreparebeforefeedingyourcells

• ChecklistforSubculturing(BectonDickinson)Thingstorememberandpreparebeforesplittingyourcellsintofreshtissueculturevessels
• HemacytometerWorkbook(BectonDickinson)Sampleproblemsfordeterminingcellnumber,celldensity,viability,etc.
• Preparingcoatedcoverslips(HoshiLab)Somecellssticktocoveslipsbetteriftheyarecoatedwithpolyaminoacidsorcollagen.Poly-(D,L,D/L)-lysineiscommonlyusedtotreatcoverslips.
• Tips(SeftonLab)Howoftendocellsneedtobetransferredandatwhatdensityshouldtheybeseededwhentheyaretransferredorpassaged?
• AdaptationofCellstoSerumFreeMedium(LTI)AdaptationofCellstoSerumFreeMedium
• Troubleshooting:CellCulture(LTI)Troubleshootingtocommonlyencounteredproblems.