用Seahorse检测线粒体功能和细胞代谢状况
来自 : 蚂蚁淘
NOTE:Thesooneraftercellfusionyoucandoalimitingdilution,thebetteryourchancesofretainingstrongpositivehybridomas.
- Removespleencellsfrom1or2mice,washasforcellfusion(above),andsUSPendthecellsinca.100mlDMEMcompletemediacontainingHT.
- Dilutehybridomacellsby1x104to1x105.Fornormalcellcultures,thiscanbeaccomplishedbyplacing0.1mlofhybridomacellsinto10mlofwashingmedia.Mix,andremove0.1mltoatubecontaining10mlofspleencellmedia(1x104dilution).Forverythicklygrowingcells,maketwodilutionsintowashingmedia(1x106dilution).
- Plate36wellsofa96welltissuecultureplatewith0.2mlofthismixture(7.2ml)(GOAL:5hybridomacells/well).Thiswillleave2.8mloftheoriginalcellsuspension.
- Totheremaining2.8mlsuspensionadd7.2mlofspleencellsuspension,andseed36wellswith0.2mlofthismixture(GOAL:1hybridomacell/well).
- Finally,add2.0mlofspleencellsuspensionandseed0.2mlintotheremaining24cells(GOAL:0.5mlhybridomacell/well).
Thefollowingstepsaretheoriginalinstructions:
- Plate36wellsofa96welltissuecultureplatewith0.1mlofthismixture(3.6ml)(GOAL:5hybridomacells/well).Thiswillleave1.4mloftheoriginalcellsuspension.
- Totheremaining1.4mlsuspensionadd3.6mlofspleencellsuspension,andseed36wellswith0.1mlofthismixture(3.6ml;1.4mlremaining)(GOAL:1hybridomacell/well).
- Finally,add1.0mlofspleencellsuspensionandseed0.1mlintotheremaining24cells(GOAL:0.5mlhybridomacell/well).)
- Dependingofcultureconditions,cloningefficiencies,counting,dilutionerror,37ofthewellsshouldhavenogrowth,asdeterminedbythePoissondistribution.
- Testthewellsthatappeartobemonoclonalforantibodyproduction(ELISA).Freezestrongpositivewells(asgivenbelow),andexpandthecelllineinvitrousingalargetissuecultureflaskorinvivobyinitiationofascitesfluidinmice(BALB/c).
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