PL_MmAtf4

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Experiments were performed in pre-sterilized polystyrene 96-square System Duetz HDW-microplates (CR1496c, Enzyscreen, Haarlem, Netherlands) capped with autoclaved low-evaporation sandwich Duetz covers (CR1296a, Enzyscreen). Cells (250 µL culture per well) were incubated in a Multitron Cell humidified incubation shaker (Infors HT, Basel, Switzerland) at following conditions: 85% humidity, 37°C, 5% CO2, 225 rpm (250 rpm when incubating transfected cells to minimize cell clumping), 50 mm orbit.	The day before transfection, anti-clumping agent was removed from cells maintained in shake flasks by two centrifugation-based (200g, room temperature [RT], 5 min) washing steps. Plasmids were diluted in OptiPROTM SFM in a V-bottomed 96-well microplate (Greiner Bio-One). One half of total DNA amount was plasmid encoding model protein and the other half was either a mock plasmid (PL_TGExpr) or plasmids encoding target genes. Subsequently, FreeStyleTM MAX Reagent was diluted in OptiPROTM SFM and immediately after mixed with diluted plasmid. After 5 minutes of complexation, plasmid:FreeStyleTM MAX reagent mix was transferred to wells in HDW-microplate containing 2.5x105 cells in 250 µL CD CHO supplemented with 8 mM L-Glutamine and 50 U/mL Penicillin-Streptomycin (Life Technologies).	3 hours post-transfection, anti-clumping agent was added to all wells reaching a final concentration of 2 µL/mL. VCD and viability were measured just before transfection (t=0) and at the following time points posttransfection: 24 h (day 1), 48 h (day 2) and 72 h (day 3). Supernatant samples were obtained by transferring cell suspension to V-bottomed 96-well microplate (Greiner Bio-One) and centrifuging (500g, RT, 5 min) the plate. Supernatants were recovered and stored at -80°C. When supernatant samples were obtained on day 2 and day 3 post-transfection (and not only on day 3), 30 µL cell suspension was diluted with 60 µL complete medium (3-fold dilution) to minimize effects of changing total cell culture volume.

Upstream tips
Experiments were performed in pre-sterilized polystyrene 96-square System Duetz HDW-microplates (CR1496c, Enzyscreen, Haarlem, Netherlands) capped with autoclaved low-evaporation sandwich Duetz covers (CR1296a, Enzyscreen). Cells (250 µL culture per well) were incubated in a Multitron Cell humidified incubation shaker (Infors HT, Basel, Switzerland) at following conditions: 85% humidity, 37°C, 5% CO2, 225 rpm (250 rpm when incubating transfected cells to minimize cell clumping), 50 mm orbit.
Protocol tips
The day before transfection, anti-clumping agent was removed from cells maintained in shake flasks by two centrifugation-based (200g, room temperature [RT], 5 min) washing steps. Plasmids were diluted in OptiPROTM SFM in a V-bottomed 96-well microplate (Greiner Bio-One). One half of total DNA amount was plasmid encoding model protein and the other half was either a mock plasmid (PL_TGExpr) or plasmids encoding target genes. Subsequently, FreeStyleTM MAX Reagent was diluted in OptiPROTM SFM and immediately after mixed with diluted plasmid. After 5 minutes of complexation, plasmid:FreeStyleTM MAX reagent mix was transferred to wells in HDW-microplate containing 2.5x105 cells in 250 µL CD CHO supplemented with 8 mM L-Glutamine and 50 U/mL Penicillin-Streptomycin (Life Technologies).
Downstream tips
3 hours post-transfection, anti-clumping agent was added to all wells reaching a final concentration of 2 µL/mL. VCD and viability were measured just before transfection (t=0) and at the following time points posttransfection: 24 h (day 1), 48 h (day 2) and 72 h (day 3). Supernatant samples were obtained by transferring cell suspension to V-bottomed 96-well microplate (Greiner Bio-One) and centrifuging (500g, RT, 5 min) the plate. Supernatants were recovered and stored at -80°C. When supernatant samples were obtained on day 2 and day 3 post-transfection (and not only on day 3), 30 µL cell suspension was diluted with 60 µL complete medium (3-fold dilution) to minimize effects of changing total cell culture volume.

Publication protocol

Experiments were performed in pre-sterilized polystyrene 96-square System Duetz HDW-microplates (CR1496c, Enzyscreen, Haarlem, Netherlands) capped with autoclaved low-evaporation sandwich Duetz covers (CR1296a, Enzyscreen). Cells (250 µL culture per well) were incubated in a Multitron Cell humidified incubation shaker (Infors HT, Basel, Switzerland) at following conditions: 85% humidity, 37°C, 5% CO2, 225 rpm (250 rpm when incubating transfected cells to minimize cell clumping), 50 mm orbit. The day before transfection, anti-clumping agent was removed from cells maintained in shake flasks by two centrifugation-based (200g, room temperature [RT], 5 min) washing steps. Plasmids were diluted in OptiPROTM SFM in a V-bottomed 96-well microplate (Greiner Bio-One). One half of total DNA amount was plasmid encoding model protein and the other half was either a mock plasmid (PL_TGExpr) or plasmids encoding target genes. Subsequently, FreeStyleTM MAX Reagent was diluted in OptiPROTM SFM and immediately after mixed with diluted plasmid. After 5 minutes of complexation, plasmid:FreeStyleTM MAX reagent mix was transferred to wells in HDW-microplate containing 2.5x105 cells in 250 µL CD CHO supplemented with 8 mM L-Glutamine and 50 U/mL Penicillin-Streptomycin (Life Technologies). 3 hours post-transfection, anti-clumping agent was added to all wells reaching a final concentration of 2 µL/mL. VCD and viability were measured just before transfection (t=0) and at the following time points posttransfection: 24 h (day 1), 48 h (day 2) and 72 h (day 3). Supernatant samples were obtained by transferring cell suspension to V-bottomed 96-well microplate (Greiner Bio-One) and centrifuging (500g, RT, 5 min) the plate. Supernatants were recovered and stored at -80°C. When supernatant samples were obtained on day 2 and day 3 post-transfection (and not only on day 3), 30 µL cell suspension was diluted with 60 µL complete medium (3-fold dilution) to minimize effects of changing total cell culture volume.

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