1. Transfer cells to a sterile tube. Centrifuge cells at 1,000 g for
5 minutes. Remove the media and resuspend the cells in fresh antibiotic-free
media. For most mammalian cells, resuspend the cells at a concentration of 2-5 x
2. Mix equal volumes of resuspended cells and 2X freezing medium (e.g., 2X MEM), place
on ice. Mix gently.
2X freezing medium (I): 15%-20% DMSO in growth media with 40% FCS and without antibiotics
2X freezing medium (II): 15%-20% glycerol in growth media with 40% FCS and without antibiotics
3. Aliquot 1 ml into cryovials. Place on ice.
4. Place vials in a prechilled styrofoam container (-70 °C) and close off completely. Or
place vials in a Nalgene Cryo Freezing Container. Place in -70 °C freezer overnight.
Cells can be stored at -70 °C for months.
5. Transfer vials to liquid nitrogen tank.