**2. Materials and methods**

#### **2.1 Cell culture**


Cells used in these studies are described in Table 1. All were grown and maintained at 37oC in 5% carbon dioxide.

\*Dulbecco's Modified Eagle's Medium

\*\*Eagle's Minimum Essential Medium

Table 1. Cell types

#### **2.2 Cell poration with H5**

The pore-forming protein H5 was obtained from the lab of Hagan Bayley (Bayley, 1994). It is derived from the bacterial toxin α-hemolysin, which forms constitutively opened pores in cell membranes. The modified bacterial toxin has been engineered to form pores in the membrane that can be opened and closed by the addition of Zn+. More specific details are presented in the discussion. Cells were plated at 10,000-20,000 cells/well the night before in 96 well microtiter plates. The next day, the cells were washed with DMEM containing 1 mM EDTA for 2 minutes and then again with DMEM to remove the EDTA. 0.2M trehalose was added and incubated for 20 minutes at 37oC followed by the appropriate concentration of H5 for the respective cell type. Cells were porated and loaded with trehalose for 1 hour at 37oC before addition of DMEM with 25 µM ZnSO4 or 10% serum to close the pores. Trehalose in DMEM was then added to the wells followed by cryopreservation using a controlled rate freezer (Planar) at ~-1.0oC/min from 4ºC to -80ºC with a programmed nucleation step at -5.0oC. Cryopreserved cells were stored overnight at <-135oC. The next day, the cells were placed at -20oC for 30 minutes followed by rapid thawing at 37oC (Campbell et al., 2003; Taylor et al., 2001). The cell cultures were washed twice and then placed at 37oC for 1 hour to recover under normothermic cell culture conditions before assessment of cell viability.
