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Star Republic: Guide for Biologists

Freezing cells

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 106 cells/ml.

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.