Editing Cell Freezing Medium (section)

== Protocol 2  ==

=== Freezing  ===

'''Preparation''' 

Cells should be healthy (&gt;90% viability) and growing in log phase. 

Sterile 1 mL cryo-vials. 

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'''Freezing''' 

Count cells in a hemocytometer. 

Spin down cells at 1000 rpm for 1 min. 

Resuspend cells in freezing medium (10% DMSO, 20% FCS, 70% media in RPMI or DMEM) at a concentration of 4 X 10e6 cells/1 mL freezing medium. 

Aliquot in cryovials 1 mL/vial. 

Place vials upright in a styrofoam box and cover well. 

Place in a -70 °C for 24 hours. 

Place vials in a wand and put in a liquid nitrogen container. 

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=== '''Thawing'''  ===

'''Preparation'''

Warm water bath to 37 °C. 

Place 5 mL media (RPMI, DMEM) in a sterile 15 mL centrifuge tube. 

Layer 2 mL FBS to the bottom of the tube, slowly, so that you can see two layers. 

Make growth media for&nbsp; new culture. For monoclonals this would be DMEM with 20% FBS and penicillin-streptomycin. 

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'''Thawing''' 

Take cells out of nitrogen storage and thaw rapidly by swirling in the 37 °C water bath. 

Sterilize the outside of the vial with 70% ethanol 

Bring in to the culture hood and add slowly to the top of the layered media. 

The cells should fall to the interface between the media and the FBS. 

Spin for 10 min at 1000 or 3000 rpm. 

Pipet out supernatant and resuspend cells in the growth media of choice. 

Pipet cells into a T25 and place in a CO2 incubator for growth.<br> 

[[Category:Protocols]]