Hello,

Suspend your leaukaemic cell containing mixture (spleen, bone marrow or PBL), centrifuge at 1200 rpm wash 2x in PBS + 1% Penicillin Streptomycin mix. When done, remove supernatant and add 500 ul Freezing Solution to each 1-100 million cells, gently resuspend the pellet and transfer to 1 ml freezing vials. Alternatively, you could directly pipette the cell slurry to the freezing vials with prefilled Freezing Solution. Freeze the cells slowly in a programmable cell freezer or put them into a styrofoam box that is in a -80/90 freezer, but roll about five layers of paper towel around each 1 ml cell freezing tube or groups of 8-12 tubes. Cells shoud be transferred to the -80/90 freezer immediately (ASAP) after contacting the Freezing Solution. 

To thaw your cells, let the cells thaw at ambient temperature for 5 minutes, then transfer them to 37 C (or hold them in your hand) until you see no ice in the vial (about 2 minutes). Gently transfer the content of the vial to a centrifuge tube, add 5-10 ml PBs/PenStrep wash 1-2x and use the cells.

Freezing Solution: 10% tissue culture quality DMSO, 90% Fetal Calf Serum (the highest quality possible).

Let me know, how it goes.

Regards,

-Laszlo

We have used mouse leukemia L5178Y cells for many years (grown both in vitro and in vivo) and kept stock frozen in liquid nitrogen (-196 deg C). The freezing protocol is to pellet the cells (at 200 g for 5-10 minutes), aspirate the medium, wash twice with PBS, and then resuspend the cell pellet in whichever growth medium you normally use for their in vitro maintenance, supplemented with 10-15% fetal bovine serum (FBS). Count the cells (Hemocytometer or electronic counter) and calculate what volume of cell mixture will be needed to get 2 million (2x10^6) cells/mL. To avoid some cells seeing a higher DMSO concentration when mixing, make medium with the same concentration of FBS (10-15%) but 20% DMSO and add an equal volume of cells in the same medium without any no DMSO, mix quickly (gentle vortex), so that the cells are quickly in medium with 10% DMSO and 10-15% FBS, at 1 million cells/mL. Dispense this solution into 2 mL cryogenic freezer tubes (about 1.5 mL each to allow space for expansion) and freeze at a cooling rate of  -1 deg C/minute until -20 deg C (or lower) is reached. We use a special freezer lid that holds vials in the vapor phase of the liquid nitrogen, at a calibrated height in the neck of the cryo-storage tank, for controlled rate freezing and calibrated the position in the neck (depth) necessary to achieve this cooling rate. Once frozen (about 1 - 2 hours, or can leave overnight), the vials can be placed in wands which are then placed in sleeves (to prevent vials from dropping out of the wands and getting lost in the liquid N2) which are then placed in the liquid nitrogen vapor (or liquid) for long term storage. If stored in liquid N2 (rather than vapor) it is essential to make sure that the vials are well sealed (one can put each vial in a sealed plastic bag to prevent possible leakage of the liquid N2 into a vial) so that they do not explode on thawing if N2 leaks into a vial. We have done this for years and successfully thawed and grown cultures stored for decades. Make sure that you top up the cryo storage tank regularly to avoid thawing of your frozen stocks.

When you want to resuscitate the cell line, take a vial and put it into a beaker containing a much larger volume of water at 37 deg C to thaw quickly. At 10^6 cells per mL, you can then readily calculate the volume of stock cell solution needed to seed new cultures at the same cell density that you normally use to culture them. You may need to try a few concentrations the first time to determine the plating efficiency, which may be somewhat lower than usual until the line is re-established.

I hope that this helps.