1.Dilute2mlof4000U/mlCollagenaseDasfollows:1mlinto9mlHBSS/Ca++/Mg++(=400U/ml)and1mlinto39mlHBSS/Ca++/Mg++(=100U/ml).Putonice.

2.Place5mlofthe100U/mlsolutionina10cmdish.Overadrydish,injectspleenswith100U/mlsolution.Holdspleenwithforcepsandpushonto22-Gneedlewhileslowlyinjecting1mlcollagenasesolution.Tearopenthespleenwiththeneedleandtransfertodishwithcollagenase.Afterinjectingallthespleens,collectanyfluidfromthedishandputinto50mltube.Rinsedishwithanother3ml100U/mlcollagenaseandalsoaddtotube.

3.Usingforceps,teasethespleensapartintosmallfragments.ThenPipettevigorouslywitha5mlpipette.Tiltthedish,andleavingthelargerfragmentsbehind,transfersolutiontotube.

4.Add10ml400U/mlcollagenasetodishwithleftoverfragmentsandincubateat37oCfor60-90minutes.Thenpipettefragmentsvigorouslyandmushwithglassslides.Rinseslideswith3ml100U/mlcollagenaseandcollectsUSPensionintotube.Rinsedishonelasttimewith3ml100U/mlcollagenaseandaddtotube.

5.Spin,10min,speed3(ca.280g).Resuspendin1mldenseBSAperspleen.Overlayupto5-6mlsplenicsuspensionin15mlconicalwith1.5mlRPMIwithnoadditions.Spin,15min,7000RPM,HS-4rotor,4oC,withslowaccelerationandnobrake.

6.TakeinterfaceplusRPMIandtransferto15mlconical.FilltubewithRPMIwithnoadditionsandmix.Spinandthendoonewash.

7.Resuspendin5mlRPMIwithnoadditions.Add0.25mlmitomycinCat0.5mg/ml.Incubate37oC,30min,thenwash3X.

Reagents:HBSS/Ca++/Mg++(Core)denseBSA(Sigma#A7409)

DawnePage,Ph.D.