p1.2-Hygro-FSH-B-chain

Protocol tips

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A DHFR-negative CHO DG-44 cell line (cat # A1097101, ThermoFisher Scientific, Waltham, MA, USA) was cultured in the shake flasks in the chemically defined suspension medium CD DG-44, supplemented with 0.18% Pluronic F-68 and 4 mM L-glutamine (ThermoFisher Scientific). The culture flasks were maintained in a humidified incubator, 37°C/ 8% CO2, on a shaker, at a constant rotation rate of 130 rpm.	The cells were passaged 24 h before transfection. Plasmids were transfected by electroporation in Gene Pulser Electroporation Buffer (Bio-Rad, Hercules, CA, USA) using a cuvette with a 4 mm gap with 7.5 million cells and 15 μg of linearized DNA for each transfection. Optimal conditions for a stable transfection were determined by dividing the transiently transfected populations into three parts, transferring them into culture medium, supplemented with 0.2; 0.5; 1 μM MTX for DG-44 cell line and 0.5; 1 or 2 μM MTX for CHO S cell line for 22 days with medium exchange every 3 days until the cell viability increased to 85%	Cells were counted by trypan blue exclusion and fluorescence microscopy at 48 h post-transfection.

Upstream tips
A DHFR-negative CHO DG-44 cell line (cat # A1097101, ThermoFisher Scientific, Waltham, MA, USA) was cultured in the shake flasks in the chemically defined suspension medium CD DG-44, supplemented with 0.18% Pluronic F-68 and 4 mM L-glutamine (ThermoFisher Scientific). The culture flasks were maintained in a humidified incubator, 37°C/ 8% CO2, on a shaker, at a constant rotation rate of 130 rpm.
Protocol tips
The cells were passaged 24 h before transfection. Plasmids were transfected by electroporation in Gene Pulser Electroporation Buffer (Bio-Rad, Hercules, CA, USA) using a cuvette with a 4 mm gap with 7.5 million cells and 15 μg of linearized DNA for each transfection. Optimal conditions for a stable transfection were determined by dividing the transiently transfected populations into three parts, transferring them into culture medium, supplemented with 0.2; 0.5; 1 μM MTX for DG-44 cell line and 0.5; 1 or 2 μM MTX for CHO S cell line for 22 days with medium exchange every 3 days until the cell viability increased to 85%
Downstream tips
Cells were counted by trypan blue exclusion and fluorescence microscopy at 48 h post-transfection.

Publication protocol

"A DHFR-negative CHO DG-44 cell line (cat # A1097101, ThermoFisher Scientific, Waltham, MA, USA) was cultured in the shake flasks in the chemically defined suspension medium CD DG-44, supplemented with 0.18% Pluronic F-68 and 4 mM L-glutamine (ThermoFisher Scientific). The culture flasks were maintained in a humidified incubator, 37°C/ 8% CO2, on a shaker, at a constant rotation rate of 130 rpm. DHFR-positive CHO S cell line (cat # R80007, ThermoFisher Scientific) was cultured in the shake flasks in the defined serum-free suspension medium, ProCHO 5 (Lonza, Basel, Switzerland), supplemented with 0.1% Anti-Clumping Agent (ThermoFisher Scientific) and 8 mM L-glutamine (ThermoFisher Scientific). The culture flasks were maintained in a humidified incubator, 37°C/ 5% CO2, on a shaker, at a constant rotation rate of 130 rpm.

The cells were passaged 24 h before transfection. Plasmids were transfected by electroporation in Gene Pulser Electroporation Buffer (Bio-Rad, Hercules, CA, USA) using a cuvette with a 4 mm gap with 7.5 million cells and 15 μg of linearized DNA for each transfection. Cells were counted by trypan blue exclusion and fluorescence microscopy at 48 h post-transfection.

Optimal conditions for a stable transfection were determined by dividing the transiently transfected populations into three parts, transferring them into culture medium, supplemented with 0.2; 0.5; 1 μM MTX for DG-44 cell line and 0.5; 1 or 2 μM MTX for CHO S cell line for 22 days with medium exchange every 3 days until the cell viability increased to 85%"

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