1)Turnonthecounterbypullingouttheon/offbutton.Youneedtodothisatleast10minbeforeusetoobtainsufficientvacuum.

Usuallyput0.2mlofyoursUSPensionofcellsinto10mlTrisorTD.Inrealityyoucanuseanydilution,aslongasyouknowhowtomultiply.2)Besuretomixthesamplethoroughlybeforecounting,sincethecellswillsettletothebottom.Tocountthesample,turntheglassvalvedirectlyabovethecellstovertical(open).Waituntilyouseeagreenbaselineinthe"sizing"monitor--thevertical"blips"representeithercellsorcrud.Thelargertheblip,thelargerthecell.TurnvalveAbacktohorizontal(closed)tobeginthecount.Thelengthofthecountshouldbeapprox.13sec,fromwhenthefirstnumberappearsintheregistertowhenthegreendisplaygoesout.Ifthecountingtimeisgreaterthan13sec,theapertureiscloggedwithdebrisandshouldbecleanedgentlywiththebrushprovided(aswellas"flushed,"see5).Anycountwhichisobtainedwhentheapertureisobstructedordownrightcloggedhasanunpredicatablecoincidenceandshouldbedoneagain.

Thecountdisplayedisthethenumberofcellsin0.5ml.Ifyoudiluted0.2mlofcellsto10ml,multiplythenumberdisplayedby100todeterminethenumberofcellspermlintheoriginalculture.Besuretousethecoincidencecorrectiontableifyourcountisgreaterthan10,000.AthighconcentrationstheprobABIlityoftwoormorecellspassingthroughtheaperturesimultaneously,andbeingcountedasonecell,ishigh.Findthefirstthreedigitsofyourcountonthetableandusethecorrectedfigure,whichisnexttoit,foryourcalculations.Obviously,ifthecellcountdisplayedistoolowortoohigh(lessthan3000orgreaterthan99,900cellsper0.5ml),youshouldsetupanappropriatedilutionandre-countthesample.

3)Whenyou"refinishedcounting,flushthesystem.PuttheelectrodeintothevialcontainingIsotergeandchecktoseethattheflasklabeled"RinseFlask"containsatleast100mlofsolution.Ifit"slow,fillitwithTrisorTDandsomeIsoterge.TurnvalveAtovertical,thenturnvalveB,whichisofftotheleft,tovertical.Flushingwilloccur.After10sec,turnvalveBbacktohorizontalandthenturnvalveAtohorizontal.StoretheelectrodeinthevialofIsoterge,and,ifit"stheendoftheday,turnthemachineoff.

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