Cell Quantification
来自 : 蚂蚁淘
AimForthemajorityofmanipulationsusingcellcultures,suchastransfections,cellfusiontechniques,cryopreservationandsubcultureroutinesitisnecessarytoquantifythenumberofcellspriortouse.Usingaconsistentnumberofcellswillmaintainoptimumgrowthandalsohelptostandardizeproceduresusingcellcultures.Thisinturngivesresultswithbetterreproducibility.
Materials
- Media–pre-warmedtoappropriatetemperature(refertotheECACCCellLineDataSheetforthecorrectmediumandtemperature)
- 70%ethanolinwater(Prod.No.R8382)
- 0.4%TrypanBlueSolution(Prod.No.T8154)
- Trypsin/EDTA(Prod.No.T4049)
Equipment
- Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
- Waterbathsettoappropriatetemperature
- MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
- Centrifuge
- CO2incubator
- Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauer,CamlabCCH.AC1)
- Invertedphasecontrastmicroscope
- Pre-labeledflasks
Procedure
- BringadherentandsemiadherentcellsintosUSPensionusingtrypsin/EDTA(Prod.No.T4049)asabove(Protocol3and4)andresuspendinavolumeoffreshmediumatleastequivalenttothevolumeoftrypsin.ForcellsthatgrowinclumpscentrifugeandresuspendinasmallvolumeandgentlyPipettetobreakupclumps.
- Understerileconditionsremove100-200uLofcellsuspension.
- AddanequalvolumeofTrypanBlue(Prod.No.T8154)(dilutionfactor=2)andmixbygentlepipetting.
- Cleanthehaemocytometer.
- Moistenthecoverslipwithwaterorexhaledbreath.Slidethecover-slipoverthechamberbackandforthusingslightpressureuntilNewton’srefractionringsappear(Newton’srefractionringsareseenasrainbow-likeringsunderthecover-slip).
- Fillbothsidesofthechamber(approx.5-10uL)withcellsuspensionandviewunderalightmicroscopeusingx20magnification.
- Countthenumberofviable(seenasbrightcells)andnon-viablecells(stainedblue)-(seebelow).Ideally>100cellsshouldbecountedinordertoincreasetheaccuracyofthecellcount(seenotesbelow).Notethenumberofsquarescountedtoobtainyourcountof>100.
- Calculatetheconcentrationofviableandnon-viablecellsandthepercentageofviablecellsusingtheequationsbelow.
Where:
- Aisthemeannumberofviablecellscounted,i.e.TotalviablecellscounteddividedbyNumberofsquares
- Bisthemeannumberofnon-viablecellcounted,i.e.Totalnon-viablecellscounteddividedbyNumberofsquares
- Cisthedilutionfactorand
- Disthecorrectionfactor(thisisprovidedbythehaemocytometermanufacturer).
Concentrationofviablecells(cells/ml)=AxCxDConcentrationofnon-viablecells(cells/ml)=BxCxDTotalnumberofviablecells=concentrationofviablecellsxvolumeTotalnumberofcells=numberofviable+numberofdeadcellsPercentageViability=(Noofviablecellsx100)dividedbyTotalNoofcells
KeyPoints
- TrypanBlue(Prod.No.T8154)istoxicandisapotentialcarcinogen.Protectiveclothing,glovesandface/eyeprotectionshouldbeworn.Donotbreathethevapor.
- Thecentralareaofthecountingchamberis1mm2.ThisareaissuBDividedinto25smallersquares(1/25mm2).Eachoftheseissurroundedbytriplelinesandisthenfurtherdividedinto16(1/400mm2).Thedepthofthechamberis0.1mm.
- Thecorrectionfactorof104converts0.1mm3to1ml(0.1mm3=1mm2x0.1mm)
- Thereareseveralsourcesofinaccuracy:
- Thepresenceofairbubblesanddebrisinthechamber.
- Overfillingthechambersuchthatsamplerunsintothechannelsortheotherchamber
- Incompletefillingofthechamber.
- Cellsnotevenlydistributedthroughoutthechamber.
- Toofewcellstocount.Thiscanbeovercomebycentrifugingthecells,resuspendinginasmallervolumeandrecounting.
- Toomanycellstocount.Thiscanbeovercomebyusingahigherdilutionfactorintrypanbluee.g.1:10
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