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Following at least 24 hours in liquid nitrogen, one vial is removed from storage and quickly thawed in a 37°C water bath until only a small frozen crystal remains.  Vials are transferred into a biosafety cabinet and diluted with an appropriate volume of DMEM/F12 + 10% FBS in a 15ml conical tube to ensure linearity with the Nexcelom Cellometer.  20µl of the cell suspension is mixed with 20µl of acridine orange/propidium iodide and counted on the Nexcelom Cellometer to determine cell counts and viability.  All cell counts are performed prior to any pelleting and washing of the samples.  These counts are estimations of the total live cell yield.

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