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The cells constitutively express the temperature-sensitive SV40 T antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This feature increases protein expression levels by permitting more plasmid copies to persist in the transiently transfected cells. Expression vectors containing the human cytomegalovirus (CMV) promoter have been shown to achieve high levels of protein expression in 293T/17 cell line.
    

Transient transfections can be performed at small and large scale. High transfection efficiencies and protein yields have been demonstrated in this cell line. ATCC recommends passaging thawed cells at least twice prior to transfection to ensure optimal viability. Prior to transfection (24 hours), seed cells at a density of 8 x 10⁵ cells/mL.

The base medium for this cell line is BalanCD HEK293 (Irvine Scientific cat# 91165). To make the complete medium, add to 475 mL of the base medium:

• 20 mL of 200 mM L-glutamine (ATCC 30-2214) for a final concentration of 8 mM
• 5 mL of ITS (Corning cat# 25-800-CR) for a final concentration of 10 µL/mL

This medium is formulated for use with a 5-8% CO₂ air atmosphere.

Subculture cells at log phase (when cells are ready for passaging, i.e., every 2-3 days, and are approximately 2 x 10⁶ cells/mL). Pre-warm fresh growth medium prior to use. Swirl the flask gently to evenly distribute cells in medium. Remove a small volume of cells from the flask and perform cell count.

1. Seed at 5x10⁵ cells/mL for a 2 day subculture and 4x10⁵ cells/mL for a 3 day subculture (weekend)

2. To maintain high cell viability, prior to seeding, centrifuge cells for 5min at 170x g

3. Discard spent media and re-suspend cell pellet in pre-warmed fresh complete growth media

4.  Pipette cells gently to break aggregates

Note: Slight aggregates may be observed, but they are easily dispersed with minimal pipetting and do not impact the performance of the cell line. Alternately, appropriate amount of fresh media maybe added directly into the flask to adjust cell seeding density. However, cell viability might be slightly compromised and decreased by 5%.