AimIngeneraltermsculturesderivedfromblood(e.g.lymphocytes)growinsUSPension.Cellsmaygrowassinglecellsorinclumps(e.g.EBVtransformedlymphoblastoidcelllines).Forthesetypesoflinessubculturebydilutionisrelativelyeasy.Butforlinesthatgrowinclumpsitmaybenecessarytobringthecellsintoasinglecellsuspensionbycentrifugationandresuspensionbypipettinginasmallervolumebeforecounting.

Materials

• Media–pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%Ethanolinwater(Prod.No.R8382)

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
• Centrifuge
• CO₂incubator
• Invertedphasecontrastmicroscope
• Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauer,CamlabCCH.AC1)
• Pre-labeledflasks

Procedure

1. Viewculturesusinganinvertedphasecontrastmicroscope.Cellsgrowinginexponentialgrowthphaseshouldbebright,roundandrefractile.Hybridomasmaybeverystickyandrequireagentleknocktotheflasktodetachthecells.EBVtransformedcellscangrowinverylargeclumpsthatareverydifficulttocountandthecenterofthelargeclumpsmaybenon-viable.
2. DonotcentrifugetosubcultureunlessthepHofthemediumisacidic(phenolred=yellow)whichindicatesthecellshaveovergrownandmaynotrecover.Ifthisisso,centrifugeat150gfor5minutes,re-seedataslightlyhighercelldensityandadd10-20%ofconditionedmedium(supernatant)tothefreshmedia.
3. Takeasmallsampleofthecellsfromthecellsuspension(100-200uL-Protocol6-CellQuantification).Calculatecells/mlandre-seedthedesirednumberofcellsintofreshlypreparedflaskswithoutcentrifugationjustbydilutingthecells.Thedatasheetwillgivetherecommendedseedingdensities.
4. Repeatthisevery2-3days.

KeyPoints

1. Ifthecelllineisahybridomaorothercelllinethatproducesasubstance(e.g.recombinantproteinorgrowthfactor)ofinterestretainthespentmediaforanalysis.

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