MATERIALS

Media:

HeatinactivatedFBS(56°Cx30min)

PBS+2%heat-inactivatedFBS

IMDM+20%heat-inactivatedFBS

Viraltransduction:Iscove"s+20%heatinactivatedFBS,

plus100ng/mLeachofSCF,IL-6,andFlt-3L,plus10ng/mLIL-11.

CFUassays:aMEM+30%FBS,1%deionizedBSA,1.2%methylcellulose,0.1mMßME.

plus3U/mLEpo,50ng/mLTpo,100ng/mLSCF,100ng/mLIL-6,?ng/mLIL-3.

AntibodiesforLineageDepletion:(Pharmingen)

T-cell:CD2IgG2b,01171D	B-cell:B220(CD45R)IgG2a,01121A
CD3IgG2b,28001D	Monocytic:Mac1(CD11b)IgG2b,01711A
CD5IgG2a,01031D	Granulocytic:Gr-1(Ly-6G)IgG2b,01211A
D8IgG2b,09821D	Eryth:Ter-119IgG2b,09081A

AntibodiesforSorting:(Pharmingenmonoclonalratanti-mouse)

Sca-1PE-IgG2a(E31-161.7)01835B	PE-IgG2aIsotypecontrol,11025A
c-Kit(2B8)FITC-IgG2b,01904D	FITC-IgG2bIsotypecontrol,11184C
FcRIIblock(clone2.4G2),01241D

Mice:

C57(Ly5.2)

C57/SJL(Ly5.1,Pep3b)

OtherReagents:

Dynabeads(Sheepanti-rat,Dynal#110.08)

Ficoll

CH296

Methylcellulose(Fisher)

DeionizedBSA(Sigma)

ProtomineSulfate

Cytokines:(PeproTech)

muSCF

huIL-6

huFlt-3L

huIL-11

Thrombopoeitin

huIL-3

Erythropoeitin(Amgen)

PROCEDURE

HarvestMarrow:1.FlushfemurswithPBS/FBSandpasscellsthrougha23Gneedle.2.AlsocollectT-cellsasaflowcytometrycontrol.3.CollectlowdensitycellsbyequilibriumcentrifugationoverFicoll-Hypaque(d=1.077g/mL).Alternatively,RBCmaybelysed.Seebloodflowcytometryprotocolfordetails.4.WashcellsinPBS/FBS.

LineageDepletion:1.ResUSPendatadensityof5x10⁷cells/mLinPBS/FBSplusantibodycocktail(eachantibodyfinaldilution=1/500v/v).2.Incubateonicefor30min.Meanwhile,washDynabeads2xinPBS/FBS.Resuspendthebeadsattheiroriginalconcentration(4x10⁸beads/mL).3.WashcellsinPBS/FBS,spin,andresuspendat10⁸cells/mL.4.Slowlyadd1vol.ofDynabeads(4beads/cell)tothecellsuspension.Incubate5min.@roomtemp.5.Exposetomagneticfieldina3mLroundbottomtubeorEppendorftubewiththecorrectsizedmagnet.Transfernon-adherentcellstoanewtube.6.Removetubewithbeadsfrommagnet.Add1/2vol.PBS/FBS,gentlymix.Exposetomagnetagainandpoolnon-adherentcellswiththefirst.7.Optional:Repeatthelineagedepletionwithanewroundofantibodyandbeads(steps1-5).8.Afterthelastmangeticbeaddepletionexposethecellstothemagnetasecondtimetoremoveanyremainingbeads.Again,saveonlythenon-adherentcells.

CellSorting(Lin-,cKit+,Sca1+):1.IncubatecellsinFcgRIIblockonicefor10min.2.StainanaliquotandwithPE-IgG2aandFITC-IgG2bisotypeantibodiesasneg.controls.IncubateremainderofcellswithSca-1PE,plusc-kitFITCantibodiesonicefor30min(allantibodies1/100v/v).4.Washwith10mLofPBS/FBS,spinandresuspendinPBS/FBSwith1µg/mLofpropidiumiodide.Filterthrough70µmnylonmesh.5.SelectcellswhicharePInegative,c-kitpositive,Sca-1positivewithintermediateforwardandside-scatter.(RunT-cellsasasizecontrol).

ViralTransduction:1.Pre-coat12wellplateswithCH-296.Toeachwelladd1.5mLofIscove"s/20%FBSplusgrowthfactors(SCF,IL-6,Flt-3L,IL-11).2.Collect5000cellsperwellinto12wellplateswith1.5mL/wellofIscove"stransductionmedia.3.Incubate48hrsat37°C,5%CO_².4.Harvestcellsbyspinningat1000rpmfor5min.5.Add0.5mLof3xgrowthfactorstoeachwell(dependingontheexperiment).6.Resuspendcellsin1mLofviralsupernatant(orcontrolmedia)andreturntowells.7.Incubate12hrsat37°C.8.Repeatviraltransduction(steps4to7).Incubateanother12hrsat37°C.9.Harvestcellsandreplatein1.5mLofIscove"s/FBSplusgrowthfactors(novirus).10.Incubate4daysat37°C.

Transplant:1.HarvestfreshcompetingmarrowfromaC57/SJL(Ly5.1)mouse.Passthrough23GneedleandresuspendinIscove"s/FBSat10⁶cellsmL.2.Harvestcells,washwithIscove"s/FBS.3.Spinat1400rpmfor5min.andresuspendin1.5mL.Recordcellcount.Add1.1mLofcellsto1.1mLofcompetingmarrowcells.TransplantintolethallyirrADIated(10Gy,⁶⁰Co)recipients.4.OfremainingcellssavesomeforFACSanalysisforGFP+lineagespecificantibodies;theremaindercanbeusedforCFUassaysinmethylcellulose.CFUassays:1.ForCFUassays:Plateanaverage20cellsin35mmdisheswith1mLaMEMand1.2%methylcelluloseplusgrowthfactors.2.Incubateat37°Cin5%CO2(plus5%O2ifavailable).3.Analyzeforerythroidcoloniesat8days(andconfirmbyBenzidinestaining).4.At12daysanalyzeforgranulocyte/macrophagecoloniesandlarge(>1.5mm)mixedcolonies(erythroid,meg,andgranulocytesconfirmedbyGiemsastaining).GFPandcellsurfaceMarkerimmunostaining:1.Incubatecellswithlineagespecificantibodiesasdescribedforcellsorting,above.IflivecellsarenotneededfixationwithdiluteformalinorparaformaldehydeisO.K.butalcoholbasedfixativesshouldbeavoidedbecausetheyallowGFPtoleakoutofcells.2.Forunconjugatedantibodies,incubatewithananti-IgG2PE-conjugatedsecondaryantibody.3.WashandincubatewithP.I.asforsorting.AnalyzeonfluorescentmicroscopeorFACSmachine.