Editing 293F Protein Production (section)

==== Cell Culture<br>  ====

1. HEK-293 cells are subcultivated every 3–4 days by inoculation in 100 mL Ex-cell® 293 medium (when used for cell culture, the medium contains L-glutamine and phenol red in a 250-mL square-shaped glass bottle at an initial cell density of 0.3 × 10^6 cells/mL.<br>2. Determine the cell density and viability by trypan blue staining using a Neubauer hemocytometer chamber and an inverted phase contrast microscope.<br>3. After cell counting, transfer 3–4 × 107 cells into a 50-mL centrifuge tube and centrifuge at 500 × g for 5 min in a standard tabletop centrifuge.<br> 

4. The medium is removed by aspiration or decanting. The cell pellet is resuspended in 10 mL of Ex-cell® 293 medium and transferred to a 250-mL square-shaped bottle containing 90 mL of prewarmed Ex-cell® 293 medium.<br>5. Attach the bottle to a platform mounted on an orbital shaker using double-sided adhesive transfer tape (3M) and agitate at 110 rpm at 37°C in a 5% CO2 atmosphere without humidity. Keep the cap of the bottle opened about one quarter of a turn.<br> 

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