CollectionofPeritonealCells
来自 : 蚂蚁淘
- Preparea10mlsyringefittedwitha26Gshortneedleandfilledwith5to7mlofmediumand2to3mlofair.Airisimportant.
- PrepareaPasteurPipettewithrubberpipetter.
- EuthanizebyCO2inhalation.AskyourComparativeMedicineDepartmentforproceduresandfacilities.Makesureyourmouseisdeadbeforestarting.
- Ifyouneedtocollectbloodfromheart,takewithalateralapproachtothethorax,soyoudon"tspillbloodintheperitoneum.
- NicktheaBDominalskinbelowthesternum,takingcarenottonicktheperitoneum.
- Grabthetwosidesofthecutandgentlyripaparttheabdominalskinwithafirmbutgentlemovement,exposingthesternumandthepelvis(Aimage).Becarefulnottoripmajorabdominalvesselsoryouwillgetbloodinperitoneum.
- Grabthesternumandgentlyfilltheabdominalcavitywiththemediumandtheair(Bimage).MakesuretokeeptheneedlefirmandsteadysoyoumakethesmallestpossIBLehole.Removetheneedle.Theperitoneumshouldself-seal.
- Holdthemouseinyourhandandshake(image)itvigorouslyaboutten-fifteentimes.Putitbackonthetable.
- Grabthesternumandsomeperitoneumwithatweezer(Cimage).Youshouldnotloosethegripuntilyouaredone.
- Withaswirlingmovementinserttheair-filledPasteurpipetteintheperitonealcavity(Cinsetimage).
- Expresstheairinthecavity,distendingtheperitoneumandstarttoaspiratethemediumandtheperitonealcells(Cimage).Yoursucessdependsofthedistensionoftheperitoneum.
- Collectallthefluidyouinserted.
- Whilestillholdingthesubsternalperitoneum,openuptheabdominalwalllongitudinally(Dimage)andcollecttheremainingfluid.
ThisprotocolhasbeenorginatedbyAlanMStallintheLeonandLenoreHerzenberg"sLabatStanford(Ref).ShakingthemousewasfoundtobenecessaryforoptimumyieldofB1cells(AMStallpersonalcommunication).
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