Cell Viability Assay细胞生存量检测
来自 : 蚂蚁淘
- acellsUSPensionismixedwithtrypanblueandexaminedbylow-powermicroscopy
- Materials
- cells
- PBS
- M3
- hemocytometer
- 0.4%trypanblueinPBS
- micropipet
- microscope
Protocol- preparecellsuspensionatahighconcentration(ca106cells/ml)
- takeacleanhemocytometerslideandfixthecoverslipinplace
- cleanthesurfaceoftheslidewith70%EtOH,takingcarenottoscratchthesemi-silveredsurface
- cleanthecoverslip,pressitdownoverthegroovesandsemi-silveredcountingarea
mixonedropofcellsuspensionwithonedropoftrypanbluecollectabout20ulintothetipofamicropipettransferimmediatelytotheedgeofthecoverslip,andletthesuspensionrunintothecountingchamber,thefluidshouldruntotheedgesofthegroovesonlyleave1-2min(donotleavelonger)placeonmicroscopeundera10Xobjectiveandfocusongridlinesinchambermovetheslidesothatthelargestareayoucanseeisboundedbythreeparallellines(1-mm2)countthecellslyingwithinthisarea.- countcellsthatlieonthetopandleft-handlinesofeachsquarebutnotthoseonthebottomorright-handlines
- hundredsofcellsper1-mm2areaisok
- iftherearelessthan100cells,countoneormoreadditionalsquares(eachsurroundedbythreeparallellines)surroundingthecentralsquare
countthenumberofstainedcellsandthetotalnumberofcellswashhemocytometerDyeexclusionviABIlitytendstooverestimateviabilityMostviabilitytestsrelyonabreakdowninmembraneintegritydeterminedbytheuptakeofadyetowhichthecellisnormallyimpermeable(e.g.,trypanblue)
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