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.