Freezing BW5147 and Hybrid T Cells for LN2 Storage

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Tony Rook
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Freezing BW5147 and Hybrid T Cells for LN2 Storage

Please find the link for the following protocol:

Freezing BW5147 and Hybrid T Cells for LN2 Storage

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sterile glass media bottle
0.45μ filters
sterile 15 ml polypropylene centrifuge tubes
Freezing medium (FM) (vol:vol:vol):
10% DMSO
20% FBS
70% DMEM

To make: mix the components for 200 ml of FM in a sterile glass media bottle (DMSO can dissolve the nitrocellulose membranes used in filters unless it is thoroughly mixed with the other components prior to filtration) and then filter through a 0.45μ filter. Aliquot at 10 ml/ tube in sterile 15 ml polypropylene centrifuge tubes and store @ -20 until needed.

Please Note: The 10% DMSO in FM is necessary for cryoprotection but is also toxic to cells at room temperature, the best results are obtained if the freezing medium and freezing
vials are kept very cold, the work is completed quickly and the cells are transferred to the freezer quickly.


1. Count the cells that are to be frozen and make sure that they are not above 1.0 X 106 per ml and that you have enough for at least one vial, 5.0 x 106 cells. Standard freezing conditions are 5.0 X 106 cells per vial in 1.0 ml FM, for most purposes the minimum number of vials of each cell line to be frozen (not
necessarily on the same day) is 3 - 4.

2. Make a chart of the cells to be frozen on a given day in your notebook with the cell counts, volume taken for freezing, total cell number frozen and number of vials to be frozen.

3. If you are freezing more that 2 cell lines list them on the chart in alpha numerical order and arrange the flasks in the same order to avoid confusion and mixing of cells.


4. Take the required amount of freezing medium from the -20 freezer, thaw in warn tap water and after thawing place on ice to chill thoroughly.

5. Label each vial on the white label area with the name of the cell line, the date and your initials, place them in the freezing block in the order that the lines are listed in your notebook.

6. Transfer the cell suspension to either 15 ml or 50 ml centrifuge tubes, depending on the volume and pellet by centrifugation for 5 min @ 1500 rpm, aspirate off the medium using a new pasteur pipette for each tube and place the tubes on ice for 5 - 10 min to chill.

7. Working quickly, resuspend the first tube of cells in the appropriate volume of freezing medium and place 1.0 ml per vial in the appropriate vial and screw the top on firmly, proceed with the remainder of the cells to be frozen in the same manner.

8. After all of the cells have dispensed, seal each vial as tightly as possible using your fingers and place in a styrofoam rack, cover with another styrofoam rack and place in the -70 freezer.

9. Keep a written record of the number of vials of cells and dates of freezing in your notebook, devote a page or two to this at the back of the notebook and reference the page in your notebook where the counts are recorded, in addition keep a record in an Excel document on the computer.

PLEASE NOTE: -70 is not low enough for long term storage and it is essential that cells be transferred to liquid nitrogen and that stocks be split between at least two LN2 storage units as insurance in case of a melt down. Cells can be transferred
the next day or up to a few weeks later, do not leave at -70 for extended periods of time. The date of transfer to LN2 should also be recorded in the notebook and in the Excel document.