Automated cell counter

In the first set of shake flask experiments, cells were seeded at 0.2 × 10⁶ cells/mL in SFM4TransFx293 medium. Once the cells reached a cell density of 1–1.5 × 10⁶ cells/mL, they were split into 50 mL aliquots in 250 mL shake flasks. Daily medium replacement (DMR) was performed for five consecutive days until induction by pelleting the cells by centrifugation (300 g, 5 min) and re-suspending them in different ratios (0–100%) of fresh versus spent medium. Cell counts and viability were determined with a Cedex Automated Cell Counter after treating the cells for 30 min with Accumax™ (Sigma–Aldrich) to reduce cell aggregation.
In the second set of experiments, cells were inoculated at a cell density of 0.3 × 10⁶ cells/mL into 100 mL of complete HyCell™ TransFx-H media supplemented with 3.5 g/L CB5 and batch cultured in a 500 mL shake flask. When cell density reached ≥5E6 cells/mL, the cell suspension was split into 20 mL aliquots in 125 mL shake flasks, and cells were induced (1 μg/mL of doxycycline and 30 μg/mL of cumate). Starting at 24 h after induction and every 24 h thereafter, cells were subjected to DMR at 50% with medium either supplemented with 3.5 g/L CB5 or not. The collected medium was filtered through a 0.45 μm filter, and LV content was titrated by GTA. Daily cell counts and viability measurements were performed using the NucleoCounter^® NC-200™.