Gibco™ Expi293F™ Cells

Catalog No.	Quantity
A14527	1 mL
A14528	6 x 1 mL

Catalog No. A14527 Supplier Gibco™ Supplier No. A14527

Description

Expi293F human cells are derived from the 293 cell line and are a core component of the Expi293 Expression System.

Expi293F human cells are derived from the 293 cell line and are a core component of the Expi293 Expression System. They are maintained in suspension culture and will grow to high density in Expi293 Expression Medium. Expi293F cells are highly transfectable and generate superior protein yields compared to standard 293 cell lines in transient protein expression.

Expi293F Cells attributes include:

• Suspension culture cells pre-adapted to Expi293 Expression Medium
• Demonstrated higher protein expression levels than other 293 cells adapted to Expi293 Expression Medium
• Exhibit more rapid cell growth and higher culture viabilities than standard suspension-adapted 293 cells
• Consistent transient expression performance of Expi293F cells for over 30 passages beyond thaw
• Also available from a cGMP bank (Cat. No. 100044202)

Frozen cells are supplied in a vial containing 1 mL of cells at 1 x 10⁷ viable cells/mL in Expi293 Expression Medium and 10% DMSO. The cells should be thawed directly into Expi293 Expression Medium. The six-vial multipack eliminates the need to expand and rebank the cells in the lab.

Cells are typically grown in flasks on a shaker platform in a humidified, CO₂ cell culture incubator. However, cultures are scalable from volumes of <1 mL in multiwell plates to >10 L in bioreactors. Protein harvest can be performed at 2–7 days post-transfection, depending on the protein expressed. For expression of recombinant antibodies, harvest is typically at 5–7 days post-transfection. There is no need for culture medium supplementation or batch feeding during expression.

Specifications

• Content And Storage: 1 x 10⁷ cells/mL, store in liquid nitrogen
• Cell Line: Expi293F
• Cell Type: Kidney (Embryonic)
• Culture Type: Suspension Cell Culture
• Form: Cryopreserved
• Species: Human
• Media Recommendation: Expi293™ Expression Medium
• No. of Cells: 1 x 10⁷
• Product Line: Expi293
• Product Type: Expi293F Cells
• Quantity: 1 mL
• Shipping Condition: Dry Ice
• Source: Human

Frequently Asked Questions (FAQs)

Is there a positive control included with the MembranePro Functional Protein Expression Kit or the Expi293 MembranePro Expression System?

No. However, you can visually tell if MembranePro particles formed via a pellet at the bottom of the tube following precipitation. You can also test the function of your MembranePro particles via receptor-ligand binding studies.

I am interested in expressing some functional membrane proteins in mammalian cells. Do you offer a system that will allow me to do this?

We offer the MembranePro Functional Protein Expression System for the expression and display of mammalian cell surface membrane proteins, including G-protein coupled receptors (GPCRs), in an aqueous-soluble format. The system uses virus-like particles (VLPs) to capture lipid raft regions of the cell’s plasma membrane as the VLPs are secreted from the cell. Using this system, it is possible to capture and display endogenous or overexpressed GPCRs and other cell surface membrane proteins in their native context for downstream assays. Since the VLPs are packaged by the cell and secreted into the culture medium, VLPs allow for the isolation of functional membrane proteins by simply decanting and clarifying the culture medium, and isolating the VLPs by precipitation. This represents a substantial savings in time, effort, and required machinery over preparing cell membrane fractions. Because VLPs capture receptor-rich regions of the plasma membrane, your GPCR may also be substantially enriched over crude membrane preparations.

The standard MembranePro Functional Protein Expression System is offered in two configurations: The MembranePro Functional Protein Expression Kit (Cat. No. A11667), which provides the convenience of a complete kit with all the reagents supplied for 10 reactions, and the MembranePro Functional Protein Support Kit, which includes the reagents for functional expression of membrane proteins but does not contain the expression vector cloning module or the 293FT host cells. The MembranePro Functional Protein Support Kit is offered in three sizes, allowing 10, 60, or 600 reactions (Cat. Nos. A11668, A11669, A11670, respectively).

For high-yield expression of functional membrane proteins in Exp293F cells, we offer the Expi293 MembranePro Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293 and the technology of MembranePro to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro Functional Protein Expression System. Please see the Application Note for more details.

Note: Expi293F cells (Cat. No. A14527) and pEF6 V5 His TOPO Expression Vector Kit (Cat. No. K961020) are not supplied with the Expi293 MembranePro Expression System and have to be purchased separately as required.

Can I leave my Expi293F culture expressing for more than 7 days?

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.

If I let my Expi293F cells grow to a higher density than recommended, will they be okay?

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.

What is the biosafety level of Expi293F Cells (Cat. No. A14527, A14528)?

Expi293F Cells is classified biosafety level 2.

As Expi293F Cells (Cat. No. A14527, A14528) are stored in a solution containing 10% DMSO, do they need to be spun down in order to remove the DMSO?

No, they do not need to be spun down to remove the DMSO. As the cells (1 mL) will be transferred to a shaker flask containing 30 mL of pre-warmed expression medium, the DMSO will be diluted so that it won't affect the cells.

Can I create a stable cell line of Expi293F Cells for antibody production using a pcDNA3.4 vector system with G418/Geneticin selection antibiotic?

Please see the following application note "Creation and Scale up of a Stable Cell Line using ExpiCHO Products" (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017764_CreatScaleup_StableCellExpiCHO_UB.pdf), that presents our suggested protocol for generation of a stable cell line from ExpiCHO-S cells, which like Expi293F, are grown in suspension.

Do I need to remove Anti-Clumping Agent and Pluronic F-68 from the medium while growing Expi293 cells? How about during transfection of these cells?

You can have Anti-Clumping Agent and Pluronic F-68 in the medium while growing Expi293 cells. You need to remove Anti-Clumping Agent at least 2 passages prior to transfection of Expi293 cells because it interferes with transfection. You can add it back to the medium after transfection. Pluoronic F-68 can be present during transfection as it does not interfere with transfection.

What method do you recommend for quantifying rabbit IgG expression from pRABBIT IgG IRES-EmGFP Positive Control Vector, Cat. No. A39243?

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.

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression with the Expi systems?

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

Do I need to change media after transfection in the Expi293 Expression System?

No. The Expi293 Expression System is designed to run without media changes. There is no need to remove transfection complexes or to change growth medium following transfection.

I only added one enhancer and not the other with the Expi293 system. Will I still get good protein expression?

The Enhancers are designed to work together for maximal expression. Addition of just one Enhancer will result in reduced expression and may be anywhere from one third to two thirds the level of expression obtained if both Enhancers had been added on time.

How soon after thawing can I use my Expi293F cells and for how long are they good to use?

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.

What method do you recommend for quantifying the rabbit Antibody-Expressing Positive Control Vector (Cat. No. A14662)?

The formation of intact IgG molecules may be quantitated using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control, 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 HRP conjugate (Cat. No. A10547), Protein A-coated plates (Cat. No. 15130 for clear plates used in colorimetric detection), TMB colorimetric substrate (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37581), and PBS or TBS buffer for washes. There is an example procedure in our Protein A-coated plates manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011310_Pierce_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note, 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.

For Research Use Only. Not for use in diagnostic procedures.