• acellsUSPensionismixedwithtrypanblueandexaminedbylow-powermicroscopy
• Materials
  • cells
  • PBS
  • M3
  • hemocytometer
  • 0.4%trypanblueinPBS
  • micropipet
  • microscope
• Protocol
  • preparecellsuspensionatahighconcentration(ca10⁶cells/ml)
  • takeacleanhemocytometerslideandfixthecoverslipinplace
    • cleanthesurfaceoftheslidewith70%EtOH,takingcarenottoscratchthesemi-silveredsurface
    • cleanthecoverslip,pressitdownoverthegroovesandsemi-silveredcountingarea
  • mixonedropofcellsuspensionwithonedropoftrypanblue
  • collectabout20ulintothetipofamicropipet
  • transferimmediatelytotheedgeofthecoverslip,andletthesuspensionrunintothecountingchamber,thefluidshouldruntotheedgesofthegroovesonly
  • leave1-2min(donotleavelonger)
  • placeonmicroscopeundera10Xobjectiveandfocusongridlinesinchamber
  • movetheslidesothatthelargestareayoucanseeisboundedbythreeparallellines(1-mm²)
  • countthecellslyingwithinthisarea.
    • countcellsthatlieonthetopandleft-handlinesofeachsquarebutnotthoseonthebottomorright-handlines
    • hundredsofcellsper1-mm²areaisok
    • iftherearelessthan100cells,countoneormoreadditionalsquares(eachsurroundedbythreeparallellines)surroundingthecentralsquare
  • countthenumberofstainedcellsandthetotalnumberofcells
  • washhemocytometer
• DyeexclusionviABIlitytendstooverestimateviability
• Mostviabilitytestsrelyonabreakdowninmembraneintegritydeterminedbytheuptakeofadyetowhichthecellisnormallyimpermeable(e.g.,trypanblue)

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