PL_Mm Ppp1r15a

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). 3 hours post-transfection, anti-clumping agent was added to all wells reaching a final concentration of 2 µL/mL.
Downstream tips
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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Papers

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Manufacturer protocol

Download the product protocol from Helene Faustrup Kildegaard, The Novo Nordisk Foundation Center f for PL_Mm Ppp1r15a below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - CHO Ppp1r15a using PL_Mm Ppp1r15a from Helene Faustrup Kildegaard, The Novo Nordisk Foundation Center f. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.