Exercise 12.7 Viability Cell Count
来自 : 蚂蚁淘
Exercise12.7-ViABIlityCellCount
Materials
- SUSPensioncultureofcells
- SteriletransferPipettes
- Stock0.2%(w/v)Trypanblue
- Hemacytometerandmicroscope
Procedure
- Gentlyswirlasuspensionculturetodistributethecellsevenly.Asepticallyremoveasmallsample(0.1ml)ofcellsfromthecultures.Placethesampleinaseparatetesttube(itneednotbesterile).
- Dilute4partsofstockTrypanBluewith1partof5Xsalineandadd0.1mlofthediluteddyetoyoursample.Mixgently.
- Setupahemocytometerandcoverslip.Immediatelyplaceadropofthestain/culturecombinationonthehemocytometer(remembertousebothsidesofthehemocytometer)andwaitoneminute.
- Observethecellswithlowpowermicroscopy.Countthetotalnumberofcells,andthenumberofstainedcells.
- Computetheconcentrationofviablecellsperml.ofculture.
Notes
TrypanBlueisastainthatisactivelyextrudedfromviablecells,butwhichreADIlyentersandstainsdeadcells.Therefore,thecellswhicharebluearedead.Thedifferencebetweenthetotalnumberofcellsandthenumberofdeadcellswouldbethenumberofviablecellsinagivenaliquotofyourculture.TrypanBlueactuallysignificantlyoverestimatesthenumberofviablecells,butissufficientforpurposesofthislab.
Approximately30%ofthecellsmeasuredasviablewithTrypanBluewillnotbeabletocontinuegrowthbeyonda24hourperiod.
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