Tissue Culture Storage of Cell Lines
来自 : 蚂蚁淘
TissueCulture-StorageofCellLinesMETHOD:- EstablishedhybridomasandothercelllinescanbestoredinliquidN2usingthismethod.
- Haveeverythingreadybeforeyoustartworkingwiththecells(i.e.coolmedia,labelvials,preparecryovials,etc.)
- Freezeonlyculturesthatarehealthyandinthemid-logphaseofgrowth(ie.,2-5x105cells/ml).
- Haveyourvialsalreadylabelledwithawaterproof,cryogenicpen.Includecelllinename,passagenumber,datefrozenandnumberofcellspervial.
- CentrifugethecellsandresUSPendin10%DMSO/90%FCSatabout1-5x106cells/ml.Thefreezingmediumshouldbe5ºC,notwarm.
- Put1mlofcellsuspensionintocryovialsandtightenthelidfingertight.Donotovertightenorthevialscouldexplodewhenthawed.
- UsetheNunccryovialswiththeO-ringandinnerthreads,nottheNalgeneones.Putthevialsinoneofthecryoracksspeciallydesignedforslow,evencooling.
- TheNalgenecryocontainerholds18vials.Thebottomisfilledwithisopropanolwhichmustbechangedafterfiveuses.Followtheinstructionsonthecontainer.
- Thebluecryorackholds50vials.Itisfilledwithwater,pluggedandplacedplugsidedown.Putthevialsrightsideupintothespacesandplacetherackinthispositioninthe-70ºCfreezer.Donotplacethemina"pre-frozenrack".Donotfill100%fullortheplugwillpopwhilefreezing.
- Recordinthecardfile:celllinefrozen;#cells;datefrozen;numberofvials;canelabel/#;whichcaneholder;yourinitials/name.
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