Expi293F™ Inducible Cells - FAQs

Expi293F细胞的生长和表达属性决定了在转染7天之后，培养基将临近耗竭，并应该已经达到最高蛋白表达量。继续孵育将导致培养细胞活力的显著下降。

尽管Expi293培养基能够支持更高的细胞密度，但我们并不推荐以超出5–6 x 10e6个细胞/毫升的密度来培养Expi293细胞，因为这会降低后续的转染和蛋白表达效率。在更高的细胞密度时，遇到培养活力降低临界点的可能性也会增加。如果您培养的细胞超出5–6 x 10e6个细胞/毫升，请按照手册中的描述传代一至两次，监控它们的活力和生长速率。使用蛋白表达对照IgG或得率已知的表达载体来测试转染效果，从而确定细胞表达性能是否受到影响。

The growth and expression characteristics of Expi293F cells are such that, by 7 days post-transfection, the culture medium should be close to being spent and maximal protein expression should have already been achieved. Continued incubation will result in a large decrease in cell culture viability.

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

While the Expi293 Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F cultures beyond 5-6 x 10e6 cells/mL, as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5-6 x 10e6 cells/mL, passage them once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression Control IgG or an expression construct of known yield to determine if cell expression performance has been impacted.

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

The Expi293F Inducible Cell Line was created using the same technology as the T-REx -293 Cell Line. Both cell lines have been created by stable transfection with the pcDNA 6/TR vector; thus, they ubiquitously express the tetracycline repressor (TetR) protein. The parental cell line of Expi293F Inducible cells is Expi293F Cells (Cat. No. A14527). The key differences between these 2 cell lines are:
- Expi293F Inducible cells are grown in suspension and are adapted to high-density culture (> 15x10E6 cells/mL).
- Expi293F Inducible cells grow in Expi293 Expression System and perform equivalently to parental Expi293F cells, thus making them ideal for high-yield, inducible protein expression.

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

The formation of intact IgG molecules may be quantified using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control vector, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. A10547), Protein A Coated Plates, Clear, 96-Well (Cat. No. 15130), TMB Substrate Kit (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37535), and PBS or TBS buffer for washes. There is an example procedure in our Protein A Coated Plates manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0011310_Thermo Scientific_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note that our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a Protein A biosensor.

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

We offer pRABBIT IgG IRES-EmGFP Positive Control Vector, Cat. No. A39243, which you can use to monitor your transfection and expression.

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

Doxycycline may be used as an alternative inducing agent in the Expi293 Inducible System. It is similar to tetracycline in its mechanism of action. Doxycycline has been shown to have a longer half-life than tetracycline (48 hr vs. 24 hr, respectively). If the use of doxycycline is desired with the Expi293 Inducible System, we recommend performing a dose-response curve to determine the optimal concentration for induction.

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

We recommend starting with a tetracycline concentration of 1 µg/mL to induce the expression of your protein of interest. However, you can modulate the level of expression by treating cells with different amounts of tetracycline. We recommend performing a dose-response curve (0-1 µg/mL; 2-fold dilution) to determine the optimal concentration of tetracycline required to induce your protein of interest.

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

We recommend using the pcDNA 5/TO Mammalian Expression Vector (Cat. No. 430096). The pcDNA 5/TO vector allows tetracycline-regulated expression of the gene of interest from the strong CMV promoter in mammalian host cells expressing the Tet repressor (TetR) from the pcDNA 6/TR vector.

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

Expi293F GnTI-, Expi293F Inducible, and Expi293F Inducible GnTI- cell lines were derived from the parental Expi293F Cells (Cat. No. A14527) and therefore, share similar morphology and growth characteristics. They are cultured in Expi293 Expression Medium and transfected using ExpiFectamine 293 Transfection Kit following our standard Expi293 protocol.

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

Expi293F cells must be recovered from freezing and passaged at least 3 times using the procedure outlined in the manual to ensure optimal performance. Cells should maintain performance for at least 30 passages if maintained in accordance with the protocol in the manual.