Method:CellCountsUsingaHemacytometer

RosalieVeile

Purpose:

Thepurposeofthisprocedureistodeterminethecelldensityoftheculture.Cellculturesalwayshavesomedeadcells;theviableandnon-viablecellscanbedistinguishedwiththeuseoftrypanbluedyeandahemacytometer.Livingcellswillnottakeupthedyewhiledeadcellsdo.

Timerequired:

5minutesfor2twocellcounts

Procedure:

1. Transfer200µlofthecellsUSPensionintoa15mlcentrifugetube.

2. Add300µlofPBSand500µloftrypanbluesolutiontothecellsuspension(creatingadilutionfactorof5)inthecentrifugetube.Mixthoroughlyandallowtostand5to15minutes.Note:Ifcellsareexposedtotrypanblueforextendedperiodsoftime,viablecellsmaybegintotakeupdyeaswellasnon-viablecells,thus,trytodocellcountswithinonehourafterdyesolutionisadded.

3. Withacover-slipinplace,useapasteurPipetteandtransferasmallamountofthetrypanblue-cellsuspensiontoachamberonthehemocytometer.Thisisdonebycarefullytouchingtheedgeofthecover-slipwiththepipettetipandallowingthechambertofillbycapillaryaction.Donotoverfillorunderfillthechambers.

4. Countallthecells(non-viablecellsstainblue,viablecellswillremainopaque)inthe1mmcentersquareandthefourcornersquares.Refertodiagrambelow.Keepaseparatecountofviableandnon-viablecells.Ifgreaterthan25%ofcellsarenon-viable,thecultureisnotbeingmaintainedontheappropriateamountofmedia;reincubatecultureandadjustthevolumeofmediaaccordingtotheconfluencyofthecellsandtheappearanceofthemedia.Aculturegrowingwellwillhavemanyclumpsofcellsandwillturnthemediafromanorangetoayellowcolorwithinseveraldays(increasetheamountofmedia).Aculturenotgrowingwellwillhavefewclumpsofcellsandthemediawillnotchangetotheyellowcolor(itmayeventurnpink,ifso,decreasethevolumeofmedia).Cellsmaybefrozenifgreaterthan75%ofthecellsareviable.Note:Ifgreaterthan10%ofthecellsappearclustered,repeatentireproceduremakingsurethecellsaredispersedbyvigorouspipettingintheoriginalcellsuspensionaswellasthetrypanbluesuspension.Iflessthan20orgreaterthan100cellsareobservedinthe25squares,repeattheprocedureadjustingtoanappropriatedilutionfactor.Repeatthecountusingtheotherchamberofthehemacytometer.

5. Eachsquareofthehemacytometer(withcoverslipinplace)representsatotalvolumeof0.1mm3or10-4cm3.Since1cm3isequivalentto1ml,thesubsequentcellconcentrationperml(andthetotalnumberofcells)willbedeterminedusingthefollowingcalculations.

   Cellsperml=theaveragecountpersquarexthedilutionfactorx104(count10squares)

   Example:Iftheaveragecountpersquareis45cellsx5x104=2,250,000or2.25x106cells/ml.

   Totalcellnumber=cellspermlxtheoriginalvolumeoffluidfromwhichcellsamplewasremoved.

   Example:2.25x106(cellperml)x10ml(originalvolume)=2.25x107totalcells.

   References:

   SigmaCatalog,1989,Commonlyusedtissueculturetechniques,p.1492.

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