Expi293F™ Cells, 6 x 1 mL - 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。

最佳的表达时间随不同的目的蛋白而发生变化，但大多数测试蛋白的最佳表达时间窗为3-7天。由于本系统具有良好的扩展性，我们推荐您可在放大扩种之前进行一个小规模测试实验，来决定何时收获目的蛋白。

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。

大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换按照手册中所描述的直接切换法或序贯切换法来操作，大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换。 注意：切换后的细胞可能无法达到与Expi293F细胞同等的高水平表达。

我们推荐使用pcDNA 3.4-TOPO TA载体（货号A14697）。这一载体含有天然的全长CMV启动子和克隆位点下游的WPRE（旱獭转录后调控元件），这两个元件均有利于基因的高水平表达（表达比pcDNA3.3 TOPO载体高大约2-3倍，而后者又比标准的pcDNA载体高2-5倍）。当然，实际表达水平还要视具体蛋白而定。 

除了Opti-MEM I低血清培养基为无血清但不是非动物源性的外，本系统中的其余成份均为非动物源性的，请参考应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf): 在非动物源性的条件下使用Expi293表达系统。

我们为蛋白的稳定高产量生产提供Freedom CHO-S与Freedom DG44试剂盒。我们为瞬时高产量生产提供ExpiCHO表达系统，Expi293表达系统，FreeStyle 293 表达系统，FreeStyle Max 293表达系统和FreeStyle Max CHO表达系统。如需在Exp293F细胞中高效表达功能性膜蛋白，我们还提供了Expi293 MembranePro 表达系统（货号A25869，A25870），这款产品将Expi293的扩展性/易用性与MembranePro技术整合起来，能够将膜蛋白的表达产量大大提升，超出标准贴壁培养的MembranePro功能性蛋白表达系统的表达产量20倍以上。请参见 应用说明(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) 以了解更多详情。

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.

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The optimal expression time will be different for each protein, but most tested proteins fall within a 3-7 day window. As the system scales well, it is recommended you run a small-scale pilot experiment to determine when to harvest your protein of interest prior to scaling up.

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.

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Other 293 cell lines may be used with the Expi293 Expression System. However, before these cell lines may be used for transfection studies, they must be adapted to serum-free, suspension culture in Expi293 Expression Medium and evaluated for transfection and expression.

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We recommend using the pcDNA 3.4-TOPO TA vector (Cat. No. A14697). This vector contains the native, full-length CMV promoter and a WPRE (Woodchuck Posttranscriptional Regulatory Element) downstream of the cloning site, both of which contribute to high level gene expression (about 2-3 fold higher expression than with pcDNA3.3 TOPO vector, which in turn provides 2-5 fold higher expression than with a standard pcDNA vector). Of course, the expression level is also protein-dependent.

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All the components of the system are animal-origin free except for the Opti-MEM I Reduced Serum Medium that is serum-free but not animal-origin free. Please see the Application Note for using the Expi293 Expression System under animal origin-free conditions:
http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf

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

For stable high-yield expression, we offer the Freedom CHO-S Kit (Cat. No. A1369601) and Freedom DG44 Kit (Cat. No. A1373701). For transient high-yield expression, we offer the ExpiCHO Expression System (Cat. No. A29133), Expi293 Expression System (Cat. No. A14635), FreeStyle 293 Expression System (Cat. No. K900001), FreeStyle Max 293 Expression System (Cat. No. K900010), and FreeStyle Max CHO Expression System (Cat. No. K900020). 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 (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) for more details.

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

Expi293F Cells is classified biosafety level 2.

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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.

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.

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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.

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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.

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