TissueCulture-StorageofCellLinesMETHOD:

1. EstablishedhybridomasandothercelllinescanbestoredinliquidN₂usingthismethod.
2. Haveeverythingreadybeforeyoustartworkingwiththecells(i.e.coolmedia,labelvials,preparecryovials,etc.)
3. Freezeonlyculturesthatarehealthyandinthemid-logphaseofgrowth(ie.,2-5x10⁵cells/ml).
4. Haveyourvialsalreadylabelledwithawaterproof,cryogenicpen.Includecelllinename,passagenumber,datefrozenandnumberofcellspervial.
5. CentrifugethecellsandresUSPendin10%DMSO/90%FCSatabout1-5x10⁶cells/ml.Thefreezingmediumshouldbe5ºC,notwarm.
6. Put1mlofcellsuspensionintocryovialsandtightenthelidfingertight.Donotovertightenorthevialscouldexplodewhenthawed.
7. UsetheNunccryovialswiththeO-ringandinnerthreads,nottheNalgeneones.Putthevialsinoneofthecryoracksspeciallydesignedforslow,evencooling.
   • TheNalgenecryocontainerholds18vials.Thebottomisfilledwithisopropanolwhichmustbechangedafterfiveuses.Followtheinstructionsonthecontainer.
   • Thebluecryorackholds50vials.Itisfilledwithwater,pluggedandplacedplugsidedown.Putthevialsrightsideupintothespacesandplacetherackinthispositioninthe-70ºCfreezer.Donotplacethemina"pre-frozenrack".Donotfill100%fullortheplugwillpopwhilefreezing.
8. Recordinthecardfile:celllinefrozen;#cells;datefrozen;numberofvials;canelabel/#;whichcaneholder;yourinitials/name.

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