Posted 15 March 2005 - 07:32 PM
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.