**2.3 Pretreatment (Incubation) with trehalose**

Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the culture medium was replaced with EMEM or DMEM containing trehalose (0-0.6M) and cultured at 37oC for varying periods of time. After culture, the solution was replaced with fresh medium containing trehalose (0-0.6M) and the cells were cryopreserved using a controlled rate freezer as described for H5 above.
