Cells plated at the appropriate density should be reaching confluence. You now have two choices: you can freeze them immediately (P0), or pass the cells 1:3 or 1:4 and then freeze them 1-2 d later when they look almost confluent (P1). Following trypsinization, cells are frozen in 1-mL aliquots in CEF media (without the pen/strep) + 12% v/v DMSO at a ratio of one 10-cm tissue-culture dish in three 1.5-mL cryovials. The cells are frozen overnight at -70°C before transferring to liquid nitrogen for long-term storage.

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