AimSomeculturesgrowasamixedpopulation(e.g.B95-8-marmoset)whereaproportionofcellsdonotattachtothetissuecultureflaskandremaininsUSPension.Thereforetomaintainthisheterogeneityboththeattachedcellsandthecellsinsuspensionmustbesubcultured.

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)
• Waterbathsetto37ºC
• MicroBIOLOGicalsafetycABInetattheappropriatecontainmentlevel
• Centrifuge
• Invertedphasecontrastmicroscope
• CO₂incubator
• Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauerGrid,CamlabCCH.AC1)
• Pre-labeledflasks
• Tissues

Procedure

1. Viewculturesusinganinvertedphasecontrastmicroscopetoassessthedegreeofconfluencyandconfirmtheabsenceofbacterialandfungalcontaminants.Givetheflaskagentleknockfirst,thismaydislodgethecellsfromtheflaskandremovetheneedforatrypsinisationstepwiththesubsequentlossofsomecellsduetothewashings.
2. Decantspentmediumintoasterilecentrifugetubeandretain.
3. WashanyremainingattachedcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)using1-2mlforeach25cm₂ofsurfacearea.Retainthewashings.
4. Pipettetrypsin/EDTA(Prod.No.T4049)ontothewashedcellmonolayerusing1mlper25cm₂ofsurfacearea.Rotateflasktocoverthemonolayerwithtrypsin.Decanttheexcesstrypsin.
5. Returnflasktoincubatorandleavefor2-10minutes.
6. Examinethecellsusinganinvertedmicroscopetoensurethatallthecellsaredetachedandfloating.Thesideoftheflasksmaybegentlytappedtoreleaseanyremainingattachedcells.
7. Transferthecellsintothecentrifugetubecontainingtheretainedspentmediumandcells.
8. Centrifugetheremainingcellsuspensionat150gfor5minutes.AlsocentrifugethewashingsfromNumber3aboveiftheycontainsignificantnumbersofcells.
9. Decantthesupernatantsandresuspendthecellpelletsinasmallvolume(10-20mls)offreshculturemedium.Poolthecellsuspensions.Countthecells.
10. Pipettetherequirednumberofcellstoanewlabeledflaskanddilutetotherequiredvolumeusingfreshmedium(refertoECACCCellLineDataSheetfortherequiredseedingdensity).
11. Repeatthisprocessevery2-3daysasnecessary.

KeyPoints

1. AlthoughmostcellswilldetachinthepresenceoftrypsinalonetheinclusionofEDTAisusedtoenhancetheactivityoftheenzyme.
2. Trypsinisinactivatedinthepresenceofserum.Therefore,itisessentialtoremovealltracesofserumfromtheculturemediumbywashingthemonolayerofcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537).Repeatedwarmingto37ºCalsoinactivatestrypsin.
3. Cellsshouldonlybeexposedtotrypsin/EDTA(Prod.No.T4049)longenoughtodetachcells.Prolongedexposurecoulddamagesurfacereceptors.Ingeneralashortertimeofexposuretotrypsinisrequiredforsemiadherentcelllines.
4. Trypsinshouldbeneutralizedwithserumpriortoseedingcellsintonewflasksotherwisecellswillnotattach.
5. TrypsinmayalsobeneutralizedbytheadditionofSoybeantrypsinInhibitor(Prod.No.T6414),whereanequalvolumeofinhibitorataconcentrationof1mg/mlisaddedtothetrypsinisedcells.Thecellsarethencentrifuged,resuspendedinfreshculturemediumandcountedasabove.
6. IfaCO₂incubatorisnotavailablegastheflasksfor1-2minuteswith5%CO₂in95%airfilteredthrougha0.25mfilter.

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