Batch Induction Experiments
The batch induction experiments were run in 24-well deep well plates (Axygen Scientific, Union City, CA) using Duetz covers (Kuhner shaker). The cultures were seeded at 2 x 105 cells/mL in 3.5 mL CD CHO medium supplemented with 100 µg/mL hygromycin B, 4.5 µg/mL bleocin and 4 mM glutamine. Cultures were incubated at 37°C, 5% CO2 and 225 rpm in a humidified incubator (Kuhner Shaker). The experiment was initiated by adding autophagy-inducing peptide (AIP) stock solution (1.65 mM) to the cultures to obtain the medium concentrations of 0, 1, 2, 3 and 4 µM. Every two days, a 300 µL sample was taken for monitoring of cell density, cell viability, cell diameter, and cell aggregation (Cedex analysis) using a Cedex automated cell counting instrument (Innovatis, Bielefeld, Germany), according to the manufacturer’s instructions. Once cultures reached a cell density above 2 x 106 cells/mL, 100 µL of the culture sample were mixed with 200 µL of 1X Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) and used for cell count and viability measurements at a 1:3 dilution. The remainder of the culture sample was centrifuged to remove the cells and approximately 150 µL of the supernatant were retained to determine the IgG production by ELISA. Each AIP medium concentration was evaluated in duplicate cultures and the entire batch induction experiment was repeated two additional times (for a total of 3 times). In addition, a batch induction experiment was performed using the negative control peptide.