Nucleocounter nc 3000 automatic cell counter

The cell line used in this work is a serum-free suspension-adapted HEK 293 cell line (HEK293SF-3F6) kindly provided by Dr. Amine Kamen from McGill University (Montreal, Canada). Cells were cultured in disposable polycarbonate 125 mL flasks with vent cap (Corning®) at 37°C, 5% of CO₂ and 85% RH at 130 rpm in a LT-X Kuhner shaker (LT-X Kuhner, Birsfelden, Switzerland). Cell culture media was HyCell™ TransFx-H media from HyClone™ (GE Healthcare, Chicago, IL, USA) supplemented with 4 mM GlutaMAX™ (Gibco, Life Technologies, ThermoFisher, San Jose, CA, USA) and 0.1% Pluronic™ F-68 Non-ionic Surfactant (Gibco, Life Technologies, ThermoFisher, San Jose, CA, USA).
Cell concentration and viability were determined using the NucleoCounter®NC-3000 automatic cell counter (Chemometec, Allerod, Denmark) according to manufacturer's instructions.
For the pseudoperfusion experiments, the total culture volume was 20 mL and media replacement (MR) was carried out centrifuging the culture at 300xg for 5 min every 12 h ensuring that the proportional volume of media was replaced depending on the condition. To maintain a MR rate of 2 reactor volume per day (RV/day), 20 mL were replaced every 12 h. For a rate of 1 RV/day, 10 mL were replaced every 12 h and for a rate of 0.5 RV/day, 5 mL were replaced every 12 h.