YFP‐TOP2β

Protocol tips

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	For transfection of production‐scale cultures, 1.2 × 109 HEK293F cells were pelleted by centrifugation, resuspended in 400 mL pre‐warmed Freestyle media in a 3 L flask with a vented cap (Genemate), and incubated for 15 min before transfection. YFP‐TOP2α plasmid DNA (1.2 mg) was added to 5 mL Freestyle media, and 1.2 mg PEI was added to 35 mL Freestyle. The two solutions were mixed together and incubated for 30 s, then added to the 400 mL culture of HEK293F cells.

Protocol tips

For transfection of production‐scale cultures, 1.2 × 109 HEK293F cells were pelleted by centrifugation, resuspended in 400 mL pre‐warmed Freestyle media in a 3 L flask with a vented cap (Genemate), and incubated for 15 min before transfection. YFP‐TOP2α plasmid DNA (1.2 mg) was added to 5 mL Freestyle media, and 1.2 mg PEI was added to 35 mL Freestyle. The two solutions were mixed together and incubated for 30 s, then added to the 400 mL culture of HEK293F cells.

Publication protocol

For transfection of production‐scale cultures, 1.2 × 109 HEK293F cells were pelleted by centrifugation, resuspended in 400 mL pre‐warmed Freestyle media in a 3 L flask with a vented cap (Genemate), and incubated for 15 min before transfection. YFP‐TOP2α plasmid DNA (1.2 mg) was added to 5 mL Freestyle media, and 1.2 mg PEI was added to 35 mL Freestyle. The two solutions were mixed together and incubated for 30 s, then added to the 400 mL culture of HEK293F cells. After 24 h, 400 mL Freestyle was added to the culture, and after 48 h another 200 mL Freestyle media was added to maintain cell growth. After 72 h, cells were pelleted by centrifugation at 400g for 10 min, resuspended in 80 mL PBS, and pelleted again at 500g before proceeding with cell lysis. For production of TOP2β (47–1212) from stable expression HEK293F cells, the desired culture volume was grown to a density of 2.5–3.0 × 109 cells per mL, then pelleted by centrifugation at 400g for 10 min, resuspended in 80 mL PBS, and pelleted again at 500g before proceeding with cell lysis.

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