1. Freshly thawn cells are split (when confluent) to as many 10-cmplates as possible.
2. When cells are confluent, trypsinize and take each plate up in 10 ml medium total.
3. Transfer to falcon tubes and spin at 1000 rpm for 10 min.
4. Remove medium and resuspend pellet in 2 ml FBS + 5% DMSO per 10 cm plate.
5. Aliquot 1 ml into Cryovial.
6. Freeze overnight in Styrofoam box or Nalgene Freezing container @ -80 deg.
7. Transfer to liquid N2 the following day (do not store at –80C for prolonged periods of time.)
8. Update the electronic file with the new stocks into the computer and print a copy of the updated file for the Tissue Culture Log binder.

EC 050413