Nucleocounter nc 250 cell counter

Cells were seeded in 250 mL Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies) and transfected with 75 μg of A1AT-Glul-ST6GAL1 plasmid or 75 μg of C1INH-Glul-ST6GAL1 plasmid (Suppl. Fig. 1) using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer's recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies. Two days after transfection, cells were transferred into 60 mL CD CHO medium lacking L-glutamine (Life Technologies) and supplemented with 1μL/mL anti-clumping agent and 0 μM, 10 μM, 30 μM or 50 μM MSX (EMD Millipore, Billerica, MA).
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). The cells were passaged in fresh selection medium every 2-3 days until viability and doubling time reached stable values. Polyclonal cell lines (pools) were seeded in duplicates at ~1 x 10 6 cells/mL with corresponding MSX concentrations. Cell densities and viabilities were determined once per day and supernatants of the pools were harvested three days after seeding and pooled within duplicates for purification of rhA1AT and rhC1INH.

For transient expression of rituximab/EPO, cells were seeded in Corning vent cap shake flasks (Sigma-Aldrich) as duplicates with cell densities ~1 x 10 6 cells/mL in 60 mL CD CHO medium supplemented with 8 mM L-glutamine (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2 and transfected with 75 μg of rituximab or EPO encoding plasmid for each flask using FreeStyle TM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to the manufacturer´s recommendations. 1μL/mL anti-clumping agent was added 24 h after transfection. pmaxGFP ® vector (Lonza) transfection was performed to measure transfection efficiencies.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). To purify rituximab and EPO, the supernatants of the transfected clones were harvested three days after transfection and pooled within duplicates.

For batch cultivation and secretome analysis, cells were seeded at 3.0 x 10 5 cells/mL in Corning vent cap shake flasks (Sigma-Aldrich, St. Louis, MI) as duplicates in 30 mL CD CHO medium supplemented with 8 mM L-glutamine and 1 μL/mL anti-clumping agent (Life Technologies). Cells were incubated in a humidified incubator at 120 rpm, 37 °C and 5% CO2.
Cell densities and viabilities were determined once per day using the NucleoCounter NC-250 Cell Counter (ChemoMetec). Secretome sample volume was calculated to harbor 20 x 10 6 cells and harvested five days after seeding to be pooled within biological replicates.