Marek: Freeze-down/Thaw

Overview
This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).

Materials
List reagents, supplies and equipment necessary to perform the protocol here.


 * 10-50 ml centrifuge tube
 * cryo tubes
 * DMSO, Tissue culture (TC)-tested (e.g. Sigma, D2650)
 * Cell culture medium (depends on cell line/cells)
 * Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
 * Freeze-down medium
 * 6-10% DMSO
 * Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
 * Basic TC centriguge (0 - 2500 rpm)
 * Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )

Freeze-down (suspension cells)
*Leave over-night at -80 gr.C. *You can store cells at -80 gr.C for up to 6 months. *Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).
 * 1) Centrifuge at 80-100 x g for 4-5 min at RT.
 * 2) Suck out the medium.
 * 3) Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
 * 4) Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.

Freeze-down (adherent cells)

 * 1) Wash cells with PBS.
 * 2) Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
 * 3) Observe cells under microscope until these are rounded.
 * 4) Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
 * 5) Centrifuge at 1000-1200 rpm for 4-5 min at RT.
 * 6) Suck out the medium.
 * 7) Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
 * 8) Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).

Thawing

 * 1) Keep frozen cells on dry ice until ready for thawing.
 * 2) Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
 * 3) Spray tube with 70% ethanol for sterilization.
 * 4) Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
 * 5) Fill centrifugation tube with culture medium: at least 10 ml total volume.
 * 6) Centrifuge for 5 min at 80-100 x g (RT).
 * 7) Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
 * 8) Optional: Exchange medium next day.

Contact

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