pGS-CD62L

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	CHO-lec3.2.8.1 cells were grown in a 100 cm2 flask until approximately 80% confluence and transfected with a mixture of 30 μg sterile-filtered pGS-CD62L plasmid DNA, 90 μL Lipofactamine 3000, 60 μL P3000 reagent, and 3 mL Opti-MEM. After 20 hours of incubation at 37 °C, cells were trypsinized, resuspended in 200 mL selection medium containing 30 μM MSX, diluted seven-fold, and plated into ten 384 well plates with 50 μL per well.	Selective medium replacements were made for wells exhibiting color change and the CD62L expressions were measured by ELISA following a manufacture’s instruction (R&D systems, Inc). The top 10% CD62L expression clones were trypsinized and transferred to 48 well plates, and their expressions were evaluated again by ELISA upon confluence.

Protocol tips
CHO-lec3.2.8.1 cells were grown in a 100 cm2 flask until approximately 80% confluence and transfected with a mixture of 30 μg sterile-filtered pGS-CD62L plasmid DNA, 90 μL Lipofactamine 3000, 60 μL P3000 reagent, and 3 mL Opti-MEM. After 20 hours of incubation at 37 °C, cells were trypsinized, resuspended in 200 mL selection medium containing 30 μM MSX, diluted seven-fold, and plated into ten 384 well plates with 50 μL per well.
Downstream tips
Selective medium replacements were made for wells exhibiting color change and the CD62L expressions were measured by ELISA following a manufacture’s instruction (R&D systems, Inc). The top 10% CD62L expression clones were trypsinized and transferred to 48 well plates, and their expressions were evaluated again by ELISA upon confluence.

Publication protocol

CHO-lec3.2.8.1 cells were grown in a 100 cm2 flask until approximately 80% confluence and transfected with a mixture of 30 μg sterile-filtered pGS-CD62L plasmid DNA, 90 μL Lipofactamine 3000, 60 μL P3000 reagent, and 3 mL Opti-MEM. After 20 hours of incubation at 37 °C, cells were trypsinized, resuspended in 200 mL selection medium containing 30 μM MSX, diluted seven-fold, and plated into ten 384 well plates with 50 μL per well. MSX concentrations were increased incrementally from 30 μM to 150 μM, then to 500 μM every two weeks. This was achieved by replacing 25 μL of selection medium in the wells with fresh selection medium containing two times the target MSX concentration. After four days of incubation in the 500 μM MSX selection medium, wells were inspected every three to four days for viable colonies. Selective medium replacements were made for wells exhibiting color change and the CD62L expressions were measured by ELISA following a manufacture’s instruction (R&D systems, Inc). The top 10% CD62L expression clones were trypsinized and transferred to 48 well plates, and their expressions were evaluated again by ELISA upon confluence. The eight best CD62L expressing clones were expanded to T-75 cm2 flasks, and the top two producers were further expanded into triple-layer flasks for recombinant CD62L production.

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