Harvesting Hematopoietic Cells from Mice 细胞培养 ...
来自 : 蚂蚁淘
- Materials
- 4micefromeachgenotype
- 4Ly5mice
- Bucketswithwetice3x
- Bucketwithdryice1x
- Dewarflaskwithliquidnitrogen
- 100mLbeakerswith95%ethanol2x
- 100mLbeakerwithdd.H2O2x
- Sterilepetridishes
- Surgicaltools
- SterileIMDMmediumaliquotedinto50mLconicaltubes
- SterileFicoll-Hypaque(d=1.077)
- Halothaneinabelljar
- Glassslides
- RBClysisbuffer
- Procedure
- Weighmouse.
- Anesthetizewithhalothaneorisoflouraneinhalationinabelljar.
- Placetheendsofsurgicalinstrumentsina100mLbeakerwithethanol.KeepasecondbeakerwithsterilewaterorPBSforwashingtheinstruments.Wettheskinwith70%ethanol.Opentheskinandpinbacktokeepthebodywallsterile.
- Openthechestandaspiratebloodfromtheheartintoa1mLsyringethrougha23Gneedle.TransferbloodtoEppendorftubewithadropofEDTA.Mixcontinuallytopreventclotting.Spot10µLontotheendofaglassslideandmakeathinsmear.StainthesmearswithHemaStainaccordingtothestandardprotocol.PerformRBCandWBCcountsonhemacytometer.Performdifferentialcountsof200WBConthebloodsmears.
- Removeandweighthymus.Placeinto1.5mLeppendorftubeandplungeintoliquidnitrogen.Storeondryiceandthenfreezeat-80C.
- Removespleenandplaceitinasterilepetridish,recordweight,keepdishonice.
- RemovefemurstakingcaretotrimoffasmuchmuscleaspossIBLe.KeepinstrumentscleanbywashinginwaterorPBSanddippingendsinto95%EtOH.
- Incellculturehood:
- Cutendsoffoffemursandflush1mLofIMDMthrougheachendintoa15mLconicaltube.
- MincespleenwithtwosterilescalpelsinafewmLofIMDM.TransfersUSPendedcellsto15mLconicaltube.
- PoolbonemarrowfromeachgenotypeandloadontoFicollintwo15mLconicaltubes.Dothesamewithspleencells.
- SpinFicolgrADIentat2500RPMat4deg.Cfor15minutes.
- Removebuffycoatbygentlypipettingandtransferittoafreshconicaltube.Q.S.to15mLwithIMDM.Add10µLto10µLtrypanblueforcellcounts.Pelletthecellsat1500RPM.Carefullyaspiratesupernatant.ResupendpelletbyflickingtubeandresuspendinIMDM.Adjustvolumesto1millioncells/mL.
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