FreezingandThawingofMammalianCellLines

Freezing

Preparation

Cellsaretobefrozeninliquidnitrogen,somakesureyourcaNISTersarerelativelyfullofnitrogenandyouhaveroom.Cellsshouldbehealthy(>90%viABIlity)andgrowinginlogphase.Youwillalsorequiresterile1mLcryo-vials;theyhaveascrew-topandrubbersealtokeepthenitrogenout.

Freezing

1. Countcellsinahemocytometer.Centrifuge10mLofcellson"2"inaclinicalcentrifugefor10minutesandresUSPendinfreezingmedium(10%DMSO,20%FCS,70%mediathatyouusedtogrowthecellssuchasRPMIorDMEM)ataconcentrationof2X10e6cells/0.5mLfreezingmedium.Aliquotincryo-vials0.5mL/vial.
2. Placevialsuprightinastyrofoamboxandcoverwell.Placeina-70°Cfor24hours.
3. Placevialsinawandandputinaliquidnitrogencontainer.

Thawing

Preparation

1. Warmwaterbathto37°C.
2. Place10mLmedia(RPMI,DMEM)inasterile15mLcentrifugetube.Layer2mLFBStothebottomofthetube,slowly,sothatyoucanseetwolayers.
3. Makeyourgrowthmediaforyournewculture.FormonoclonalsthiswouldbeDMEMwith20%FBSandpenicillin-streptomycin.

Thawing

1. Takeyourcellsoutofnitrogenstorageandthawrapidlybyswirlinginthe37°Cwaterbath.
2. Sterilizetheoutsideofthevialwith70%ethanol,bringintotheculturehoodandaddslowlytothetopofthelayeredmediayouprepared.ThecellsshouldfalltotheinterfacebetweenthemediaandtheFBS.
3. Centrifugeon"3"for10min.inaclinicalcentrifuge.
4. Pipetofthesupernatantandresuspendinthegrowthmediaofchoiceyoupreparedearlier.PipetintoaT25andplaceinaCO2incubatorforgrowthofyournewculture.

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