freezing of cells - (Mar/14/2005 )
my protocol says this:
1. spin down the remaining cells.
2. resuspend cells with 1.5ml medium.
3. add 1.5ml 2X freezing medium.
4. freeze down cells in 1.6ml cryovials.
my question; why should i add 1.5ml medium (step 2) when my vial only has a total volume of 1.6ml. should i ignore step 2? any ideas? 
thanks.
well, whatever your "2x freezing medium" consists off, it obviously has twice the concentration you will finally need.
that´s why you mix 1,5ml of cells resuspended in normal medium with the same volume of 2x freezing medium.
what about using two cryovials?? 
hi
i resspen cels in 1ml of serum+10%DMSO mixture or 1ml of serum free medium +10%DMSO mixture.
But in your case that means that your cells should be divided in two vials. I suppose this protocol is for freezing cells that are 80% confluent or more. So in order to prevent a passage of cells when thawing them, and by the way damage half of the quantity, passage is done before freezing.
fred
What do you mean with passage? Can you explain what you mean? Thank you very much.
hi
i'm not very ok with this term.
Wat i meant in my reply was that when cells need to be divided (due to a high confluency) it's more suitable (for cells and for your cell-stock tat you divise the quantity in two and then freeze instead of freezing all cells in the same vial and make the division after thawing.
i hope i've cleared my topic...
fred
thanks.
Hi
Most protocols say to freeze cells at 3*10 to the 7 cells per mL, this is standard across most cell lines and cell types.
If you are having trouble with fitting all of your cell suspension into one tube, then try using a bigger tube or aliquoting into more than one tube!
Routinely you need to add your standard media to resuspend the cells (step two in the original question) and dilute your freezing medium in this, dropwise with mixing, to a 1X concentration. If you do not resuspend your cells in standard media, and just go straight to the freezing medium, then the cells will die, typically because the freezing medium contains DMSO or some similar component that is actually toxic to the cells, but protects them from ice crystal formation when freezing occurs.
you meant 3x10^7 cells/ml?
1. spin down the remaining cells.
2. resuspend cells with 1.5ml medium.
3. add 1.5ml 2X freezing medium.
4. freeze down cells in 1.6ml cryovials.
my question; why should i add 1.5ml medium (step 2) when my vial only has a total volume of 1.6ml. should i ignore step 2? any ideas?
thanks.
I would recommend you use Recovery™ Cell Culture Freezing Medium (Invitrogen, Cat #:12648-010). It contains Dulbecco`s Modified Eagle Medium (D-MEM), fetal bovine serum, bovine serum, and 10% DMSO. It makes life much easier!
As a cell culturist for 18 years I can tell you
1) 3x10e7 cells/ml is a lot of cells to freeze in one hit and not necessary unless the cell line doesn't like lower cell numbers. For most non-adherent cells, I use 0.5-1x10e7 cells/ml and adherent cells I can go down to 2.5x10e6 cells/ml.
2) Commercial freezing-medias are not necessary unless the cell line is fragile (primary cells are a prime example). Most cell lines after pelleting can be resuspended directly in media+10% DMSO at RT and then aliquoted into the number of vials required. The only cells that I have worked with that can't take DMSO directly are primary white blood cells, where we add the DMSO last, while the cells are on ice.
But back to the original question from Indoubt, the answer is use 2 cryovials.