Freezing and Thawing of Mammalian Cell Lines
来自 : 蚂蚁淘
FreezingandThawingofMammalianCellLines
Forlongtermstorageofmyelomacells,hybridomacells,Tcells,andothermammaliancelllinesinliquidnitrogen,andrestoringtheminculture.
Freezing
Preparation
Cellsaretobefrozeninliquidnitrogen,somakesureyourcaNISTersarerelativelyfullofnitrogenandyouhaveroom.Cellsshouldbehealthy(>90%viABIlity)andgrowinginlogphase.Youwillalsorequiresterile1mLcryo-vials;theyhaveascrew-topandrubbersealtokeepthenitrogenout.
Freezing
- Countcellsinahemocytometer.Centrifuge10mLofcellson"2"inaclinicalcentrifugefor10minutesandresUSPendinfreezingmedium(10%DMSO,20%FCS,70%mediathatyouusedtogrowthecellssuchasRPMIorDMEM)ataconcentrationof2X10e6cells/0.5mLfreezingmedium.Aliquotincryo-vials0.5mL/vial.
- Placevialsuprightinastyrofoamboxandcoverwell.Placeina-70°Cfor24hours.
- Placevialsinawandandputinaliquidnitrogencontainer.
Thawing
Preparation
- Warmwaterbathto37°C.
- Place10mLmedia(RPMI,DMEM)inasterile15mLcentrifugetube.Layer2mLFBStothebottomofthetube,slowly,sothatyoucanseetwolayers.
- Makeyourgrowthmediaforyournewculture.FormonoclonalsthiswouldbeDMEMwith20%FBSandpenicillin-streptomycin.
Thawing
- Takeyourcellsoutofnitrogenstorageandthawrapidlybyswirlinginthe37°Cwaterbath.
- Sterilizetheoutsideofthevialwith70%ethanol,bringintotheculturehoodandaddslowlytothetopofthelayeredmediayouprepared.ThecellsshouldfalltotheinterfacebetweenthemediaandtheFBS.
- Centrifugeon"3"for10min.inaclinicalcentrifuge.
- Pipetofthesupernatantandresuspendinthegrowthmediaofchoiceyoupreparedearlier.PipetintoaT25andplaceinaCO2incubatorforgrowthofyournewculture.
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