Hi,
I am having a stable clone having G418 resistance and puromycin resistance. After expanding the cells, I freezed using 50% FBS, 40% medium and 10% DMSO, left the cells in -80 and then froze in liq nitrogen. But when I revived the cells and added the medium with the antibiotics, almost 95% of the cells were floating the next day without getting attached. Did I d something wrong in the procedure?. Is it ok if I add some antibiotic in the freezing medium ( G418 and puro). Expert advise needed.
Try freezing your cells in 90%FCS and 10% DMSO, did you use a freezing pot when placing cells in -80, or did you put the vials in a box without any form of insulation.
HI,
the question is if cell without stable construct also die. Then it would be a problem of your freezing/thawinf strategy. Further more I never added antibiotics to freezing media. When cellsare thawed they are already more fragile compared to a growing culture. Therefore I would add selection pressure 24-48 h after thawing. Especial Puro is very harsh and you can also think about reducing concentraiton
Hi Jaya, just to add to the answers above, use 90% FBS and 10% Tissue Culture Tested DMSO.
Off the shelf DMSO can have many toxic agents.
Also, remember that you need to freeze the cells slowly at -80. I wrap the cell vials that I want to freeze in multiple layers of tissues-kinda like a mummy before putting them in the -80.
Don't keep them for more than a week at -80. Transfer them to LN2 as soon as you can.
Additionally, when you thaw out the cells, you have to do it fast! Take out a vial and swirl it by hand in a 37 water bath. Do not leave it sitting in at 37!.
When the contents of the vial is liquid, quickly add the contents to a flask (or a dish)containing media that has ~10X volume of the vial. This will slowly dilute out the cellular DMSO and allow the cells to adjust to the new conditions.
May be there is some mistake in the freezing the sample, I will try it slowly. Thank you
I think that would help, the ideal condition for freezing cells is at 1 degree per min, This is achieved either by using some form of insulation as Steingrimur suggests or you can buy freezing pot called Mr frosty cat no 5100-0001.
ok. I will check the availability for it in the institution. Thank you
It would be nice to know the type of cells you are freezing. This would led you even to different freezing strategies.
let me know
Best
Jose A.
Hey in case you don't have access to mr. frosties you can use polystyrene e.g. the plates cuvettes for photomoeters were delivered with. You take the cryo tupe, wrap it with paper towl and put in one of this polystyrene slots.
further more it is of interest which cell type you freeze and if the cells are potential sensitive for DMSO. What is the recommedation from your supplier?
Regards
Sven
The cells which I use is preosteoblast and mesenchymal cells, I put the cells in polystyrene box, and place it in -80 and after 2 days, I place in liq Nitrogen. The cells are very precious to me, because its a stable clone expressing 4 proteins of interest.
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