Generally I use complete growth medium (EMEM) + 10 % FBS +5 % DMSO and freeze them at -80 °C. However it is advised to keep them in liquid nitrogen for a very long storage. You can go through the attachment.
I freeze my Caco in 90%FBS and 10%DMSO using a Cool Cell, in a -80oC freezer, this tub freezes cells -1oC/min. After around 2hr or O/N, i transfer them to Liquid Nitrogen. Thaw out with a 80-90% recovery everytime.
We freeze CaCo2 (and many other cell lines) in DMEM+25% FCS+10% DMSO (tissue culture grade) using a Cool Cell in -80C freezer.
As per my experience, I always used DMEM-F12 + 10% FBS +10%DMSO and keep it in a thermocol box for overnight/ 2 to 3 days and then transfer to liquid nitrogen..
Keynote, when you split cells, that time use less volume of trypsin/ i usually add 1ml trypsin in 10cm dish then immediately suck out all and put it into CO2 incubator for 3min and then do above steps...
Best Wishes...
Good Luck...
Hello!
For stocking any type of cells line, remember that always store 10^6 cells/cryo tube, it maximize the viability of cells; cells grow with no problem whenever you want to culture. Use the freezing media by Gibco. when yoy add freezing media to cryo tube (1ml), store it for 30 min in freezer, after that 24 hrs at -80°, then store in liquid nitrogen.
Hope it will help.
Remeber also to store your cells at an early number of passages. When I receive new cells I always prepare stocks for cryo preservation in the following days. In case of problem you can always re-start your experiments.
We freeze them in DMEM, 30% FCS, 10% DMSO. Over night in a CoolCell (or if not available, a thick layer of cotton wool does the job, too) and then transfer to liquid nitrogen. Never had problems with viability or cell growth after thawing.
Thank you Maurizio and Marianne. important points to note indeed...
Thank you so much...
best wishes..
I freeze CaCO2 and other cell lines which i work using 90% FBS and 10% DMSO (sterile) (cryo medium) mixture. Use large number of cells better use earlier passages cells and don't leave the cells longer time in the cryo medium which will may kill the cells. leave the cells in -80 C for overnight, careful before u put the cells in to the -80 C vials should not expose directly it should cover properly like keep inside the Styrofoam box and next day store in liquid nitrogen for long time storage. All the best
I would suggest to use FBS with 10% DMSO and try also with culture medium with 10% DMSO but FBS should do the work effienctly. I would suggest to use Mr. Frosty™ Freezing Container, to achieve a rate of cooling very close to -1°C/minute, the optimal rate for cell preservation. http://www.thermoscientific.com/content/tfs/en/product/mr-frosty-freezing-container.html but in case you don´t have something like this, you can freeze your samples at -20, transfer them to -80 and then to liquid nitrogen. Good luck!
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