FreeStyle™ 293 Expression System

Culture viability should be in the high 90% range for the seed stock that you are maintaining and growing for the transfections. After transfection, the viability will slowly decrease to the lower 90% to 80% range by about 3-6 days post-transfection and can drop more dramatically at 7 days or longer. This is when using our recommended protocol, which does not suggest any media changes or supplementation post-transfection.

A few parameters we recommend checking to assure high culture viability are: pH at ˜7.0, no visible clumping of the cells, CO2 levels at approximately 7-8% (this should be adjusted to achieve the appropriate pH), volume of culture at 40% or less of nominal flask capacity, and incubator temperature at 37 degrees C. If you have a shaker platform setup in your incubator, the platform may generate sufficient heat to increase the temperature of the incubator. We recommend using a thermometer attached to the shaker platform. Use of baffled flasks may help, if insufficient oxygenation of the cultures is suspected. We also recommend handling and storing the culture media properly, as light exposure can result in breakdown of certain media components. If you are using FreeStyle 293 Expression Medium, supplementing with Pluronic reagent is not necessary.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

The two systems have the same components except for the transfection reagent and the medium supplied for formation of transfection complexes. The FreeStyle Max 293 Expression System contains FreeStyle Max Reagent and OptiPRO SFM (serum-free medium that is devoid of animal-origin components), whereas the FreeStyle 293 Expression System contains 293fectin Transfection Reagent and Opti-MEM I Reduced Serum Medium (serum-free medium but not animal-origin free).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Generally, any mammalian expression vector should be compatible with the FreeStyle expression system. Some of the more popular choices include pcDNA3.3-TOPO TA vector (Cat. No. K8300-01), pcDNA3.4-TOPO TA vector (Cat. no. A14697), pcDNA3.2 DEST vector (Cat. No. 12489018), pCEP vector (Cat. No. V044-50), or pcDNA4/HisMax vector (Cat. No. K864-20).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

FreeStyle F17 Expression Medium was designed from a different base formulation than FreeStyle 293 Expression Medium. FreeStyle F17 medium shows equal or better performance than the FreeStye 293 medium for certain cell lines and proteins. It is capable of supporting the growth of 293 and CHO cells and offers a wider variety and selection of transient cell culture media options.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.