PreparationofFeederLayersAndSNLStocks1.1.GelatinizingPlatesPrepareplatesbycoveringsurfacewith0.1%Gelatinsolution:PlateSize(cm)AmountofGelatin(ml)32641091518Swirltheplatestocovertheentiresurface.Allowplatestogel@roomtemp.foratleast2hours.Aspirateoffallofthegelatindirectlybeforeusingtheplates.1.2.FeederLayersUsethe15cmstockplatestomakefeeders.1.Remove12mlsofthemediafromeachplate.2.Add360ulMitomycinCstocksolutiontoeachplateandswirltocoversurface.3.Incubate@37oCfor2hours.4.Afterincubation,aspirateoffallofthemediaandwashthecellstwicewithPBS,5-10mlperwash(enoughtocoverthesurface).AspirateoffallofthePBS.5.Add2mlofTrypsintoeach15cmplate.Swirltocovertheentiresurface.Incubate@37oCfor5minutes.6.Add5mlofmedia(7%FCS,1XGPS)toeachplatetoinactivatethetrypsin.SUSPendthecellsbypipettingup-and-downandtransferthesuspensiontosterile15mlcentrifugetubes.Repeatthesuspensionprocess,poolingallofthesuspensionsintothe15mltubes,untilallofthecellshavebeenharvested.(Youshouldbeabletopoolsuspensionsfromtwo15cmplatesintoone15mltube.)7.Spinthetubesfor7minutes@1,000rpm.8.Carefullyaspirateoffthesupernatant.Donottouchthecellpellet.Tapeachtubegentlytobreakupthepellet.9.Add5mlofmediatoeachtubeandresuspendthecellpellets.VigorouslyPipetteup-and-downtoinsureagoodsuspension.Finally,poolallofthesuspensionsintoone50mltube.Itisimperativethatthetotalvolumeofmediaaddedtoresuspendthecellsberecorded;thatvaluewillbeusedlaterincalculations.10.Transfer200ulofthecellsuspensiontoaplasticvialandadd10mlPBSorIsotonII.CountthecellsusingtheCoulterCounter.CalculatingtheCellNumber(onlyrelevantifthecounterissettocounta0.5mlsample):TheCoulterCounterwilldisplay"XXXXX",which="X,XXX,X00cells/ml"="X.Xx106cells/ml"Tocalculatethetotalnumberofcells,multiply"X.Xx106cells/ml"bythevolumefromwhichthe200ulaliquotwastaken(seestep9).11.Werequire3.5x105cells/ml.Therefore,inordertoreachthiscelldensity,youmustcalculatethevolumeofmediaintowhichthecellswillbeadded.Todothis,dividethetotalcellnumberby3.5x105cells/ml.Example:Aftercentrifugingmycellsandaspiratingoffthemedia,Ihave3cellpelletstoresuspend.Therefore,Iuseatotalof15mlofmediatoresuspendthe3pellets.Ithencounta200ulaliquotofthesuspension,andtheCoulterCounterdisplays69579=6,957,900cells/ml=(6.9x106cells/ml)x15ml(totalvolumeusedtoresuspendcells)=(1.035x108cellstotal)/(3.5x105cells/ml)approx.=296mlmedianeeded12.Onceyouhavedeterminedtherequiredvolumeofmedia,addthecellsuspensiontothatvolumeofmediaandshaketouniformlydistributethecells.13.Checktoseeifthepropercelldensityhasbeenreachedbytakinga200ulaliquotofthemediaandcountingasbefore.However,whencalculatingthetotalcellnumber,besuretousethetotalvolumeofmediatowhichthecellswereadded.Example:Fromthepreviousexample,wecalculatedthat296mlofmediawasneededtoreachtherequiredcelldensity.Uponrecountingtocheckthedensity,theCoulterCounterdisplayed04358=435,800cells/ml=(0.43x106cells/ml)x296ml(totalvolumetowhichcellswereadded)=(1.2728x108cellstotal)/(3.5x105cells/ml)approx.=364mlmedianeededIfthetotalcellcountisstilltoohightogivethedesireddensity,addthedeterminedamountofmediathatwilldilutethecellstotheproperdensity(i.e.,thedifferencebetweenthevolumeofmedianeededforthefirstcountandthevolumeneededforthesecondcount).Example:Fromtheseconddeterminationabove,wefoundthat364mlofmediaisneededtoobtainthedesiredcelldensity.Sincewehavealreadyadded296mlofmedia,weneedonlyaddanother68ml.364ml-296ml=68mlofmediatobeadded14.Oncetherequiredcelldensity(3.5x105cells/ml)hasbeenreached,aliquotthepreparedFeederMediaintothesizeandnumbersoffeederplatesneeded(rememberingtouseplatesthathavebeenpretreatedwiththegelatin).Swirltheplatestouniformlydistributethecells.SizeofPlate(cm)AmountofMediatoAliquot(ml)15301012643(or6-well)224-or4-well0.515.Placethefeedersinthe37oCincubator,labellingthetraywith"Feeders"andthedate.SNL(STO-Neo-LIF)StocksUseaconfluent10cmplatetopreparestockplates.Gel15cmplatesforthestocksandone10cmplateforthepassageplate.1.AspirateoffallofthemediaandwashthecellstwicewithPBS,4-7mlperwash(enoughtocoverthesurface).AspirateoffallofthePBS.2.Add0.8mlTrypsintotheplate;swirltocovertheentiresurface.Incubate@37oCfor5minutes.3.Add10mlofmediatotheplatetoinactivatethetrypsin.Vigorouslypipetteup-and-downtosuspendthecells.4.Determinethetotalvolumeofmediathatwillbeneededforthestockplatesbasedonthefollowing:SizeofPlate(cm)AmountofMediatoAliquot(ml)153010125.Addthecellsuspensiontotherequiredvolumeofmedia.Shakethebottletoinsureauniformdistributionofcells.6.AliquottheSTO-NeoMediaintoplatesinthevolumespecifiedforeachsizeofplate(seestep4).Remembertouseplatesthathavebeenpretreatedwithgelatin.7.Placethestockplatesinthe37oCincubator,labellingthetraywiththeclonenumber(e.g.,SNL76/7),thepassagenumber,andthedate.NOTE:The15cmplateswillbeuseduponconfluencetopreparethefeederplates.Theone10cmplatewillbeusedtomaintainthecelllinethroughanumberofpassages.FromtheLaboratoryofDr.AllanBradleyBaylorCollegeofMedicine,Houston,Texas