AimAdherentcelllineswillgrowinvitrountiltheyhavecoveredthesurfaceareaavailableorthemediumisdepletedofnutrients.Atthispointthecelllinesshouldbesub-culturedinordertopreventtheculturedying.TosubculturethecellstheyneedtobebroughtintosUSPension.Thedegreeofadhesionvariesfromcelllinetocelllinebutinthemajorityofcasesproteases,e.g.trypsin,areusedtoreleasethecellsfromtheflask.However,thismaynotbeappropriateforsomelineswhereexposuretoproteasesisharmfulorwheretheenzymesusedremovemembraneMarkers/receptorsofinterest.Inthesecasescellsshouldbebroughtintosuspensionintoasmallvolumeofmediummechanicallywiththeaidofcellscrapers.

Materials

• Media–pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%ethanolinwater(Prod.No.R8382)
• PBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)
• 0.25%trypsin/EDTAinHBSS,withoutCa₂₊/Mg₂₊(Prod.No.T4049)
• Trypsin(Prod.No.T4424)
• SoybeantrypsinInhibitor(Prod.No.T6414)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat,safetyvisor)
• Waterbathsettoappropriatetemperature
• MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
• CO₂incubator
• Pre-labeledflasks
• MarkerPen
• Pipettes
• AmpuleRack
• Tissue

Procedure

1. Viewculturesusinganinvertedmicroscopetoassessthedegreeofconfluencyandconfirmtheabsenceofbacterialandfungalcontaminants.
2. Removespentmedium.
3. WashthecellmonolayerwithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)usingavolumeequivalenttohalfthevolumeofculturemedium.Repeatthiswashstepifthecellsareknowntoadherestrongly.
4. Pipettetrypsin/EDTA(Prod.No.T4049)ontothewashedcellmonolayerusing1mlper25cm₂ofsurfacearea.Rotateflasktocoverthemonolayerwithtrypsin.Decanttheexcesstrypsin.
5. Returnflasktotheincubatorandleavefor2-10minutes.
6. Examinethecellsusinganinvertedmicroscopetoensurethatallthecellsaredetachedandfloating.Thesideoftheflasksmaybegentlytappedtoreleaseanyremainingattachedcells.
7. Resuspendthecellsinasmallvolumeoffreshserum-containingmediumtoinactivatethetrypsin.Remove100-200uLandperformacellcount(Protocol6-CellQuantification).
8. Transfertherequirednumberofcellstoanewlabeledflaskcontainingpre-warmedmedium(refertoECACCCellLineDataSheetfortherequiredseedingdensity).
9. Incubateasappropriateforthecellline.
10. Repeatthisprocessasdemandedbythegrowthcharacteristicsofthecellline.

KeyPoints

1. Somecultureswhilstgrowingasattachedlinesadhereonlylightlytotheflask,thusitisimportanttoensurethattheculturemediumisretainedandtheflasksarehandledwithcaretopreventthecellsdetachingprematurely.
2. AlthoughmostcellswilldetachinthepresenceoftrypsinalonetheEDTAisaddedtoenhancetheactivityoftheenzyme.
3. Trypsinisinactivatedinthepresenceofserum.Therefore,itisessentialtoremovealltracesofserumfromtheculturemediumbywashingthemonolayerofcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537).
4. Cellsshouldonlybeexposedtotrypsin/EDTA(Prod.No.T4049)longenoughtodetachcells.Prolongedexposurecoulddamagesurfacereceptors.
5. Trypsinshouldbeneutralizedwithserumpriortoseedingcellsintonewflasksotherwisecellswillnotattach.
6. Trypsinmayalsobeneutralizedbytheadditionofsoybeantrypsininhibitor(Prod.No.T6414),whereanequalvolumeofinhibitorataconcentrationof1mg/mlisaddedtothetrypsinisedcells.Thecellsarethencentrifuged,resuspendedinfreshculturemediumandcountedasabove.Thisisespeciallynecessaryforserum-freecellculture.
7. IfaCO₂incubatorisnotavailablegastheflasksfor1-2minwith5%CO₂in95%airfilteredthrougha0.25mfilter.

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