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.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。