Editing 293F Expression (section)

== Transient Transfection of HEK-293F Suspension Cultures using PEI ==

Cells are grown in suspension on a platform shaker in a humidified 37°C CO2 incubator with rotation at ~150 rpm. Maintain cultures for 5-7 passages prior to performing transfections to ensure stable growth patterns. When using suspension-adapted HEK293 cells (FreestyleTM 293-F cells, Life Technologies, Cat. No. R790-07) grow the cultures in FreestyleTM 293 Expression Medium (Life Technologies, Cat. No. 12338026). Cells should be maintained between 4 x 10<sup>5</sup> and 3 x 10<sup>6</sup> cells/ml in a volume not to exceed 20% of the total volume of the culture flask. Optimum rotation rate should be determined for each flask type and culture volume. FreestyleTM 293-F cells from Life Technologies are prone to clumping at higher densities and volumes.

'''Polyethylenimine (PEI)''' (25 kDa linear PEI, Polysciences, Inc., cat. No. 23966) is prepared as a stock solution at a concentration of 1 mg/ml in a buffer containing 25mM HEPES and 150 mM NaCl (pH 7.5). The PEI is added to the buffer and vortexed until completely dissolved (this can take MANY minutes of vortexing). Once fully dissolved PEI can be sterile filtered using a 0.22 μM syringe filter, aliquoted, and frozen at -20°C until needed. 

For best transfection efficiency, cells should have a viability of &gt;95% at the time of transfection. Twenty four hours prior to transfection, split cells to a density of ~1 x 106 cells/ml and culture overnight in the CO2 incubator with shaking at 37°C. Cell density should be ~2 x 10<sup>6</sup> at the time of transfection.

Transfections should be performed at a cell density of between 2.5 x 10<sup>6</sup> and 3.0 x 10<sup>6</sup> cells/ml. Count cells using a hemocytometer and spin down a sufficient volume to resuspend the cells at a density of 2.5 x 10<sup>6</sup> cells/ml in fresh 293F Freestyle Media in the desired volume for transfection. Resuspension in fresh media prior to transfection is critical. Conditioned media contains metabolites that inhibit transfection. Cells should be spun at 1200 rpm for 10 min at room temperature. Transfections will be diluted 1:1, 24 hours after transfection, so the initial volume transfected is one half of the final volume desired.

Example: culture density of the suspension culture stock = 2.8 x 10<sup>6</sup> cells/ml.<br>For a final transfection volume of 100 ml, you will need 50 ml of cells at a density of 2.5 x 10<sup>6</sup> cells/ml.<br>V1 · 2.8 x 10<sup>6</sup> cells/ml = 50 ml · 2.5 x10<sup>6</sup> cells/ml<br>V1 = 44.6 ml<br>Spin 44.6 ml at 1200 rpm for 10 min and remove media from cell pellet.<br>Resuspend cells in 50 ml of fresh pre-warmed FreestyleTM 293 medium.<br>Return the culture to the CO2 incubator while preparing DNA and PEI mixtures for transfection. Reserve an aliquot of the culture for cell counting to confirm cell density.