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Comparison of luciferase expression level in Expi293F™ Cells

This figure compares transfection reagent performance in Expi293F™ cells. Cells were transfected with a secreted Luc expression vector using commercially available transfection reagents and cultured in 30 mL of Expi293™ Expression Medium. 96 hours after transfection, cell supernatants were measured for emission intensity by Luciferase assay.

Expi293™, Freestyle™, Expi293F™ are registered trademarks of Thermo Fisher Scientific.

High production yield for IgG in Expi293F™ Cells

Heavy and light chains expression vectors were transfected using commercially available transfection reagent in Expi293F™ Cells. The Expi293F™ Cells were cultured in 30 mL of Expi293™ Expression Medium. 120 hours after transfection, supernatants were collected and electrophoresed. IgG was measured by CBB staining.

Comparison of luciferase expression level in FreeStyle™ 293-F Cells

This figure compares transfection reagent performance in FreeStyle™ 293-F Cells. Cells were transfected with a secreted Luc expression vector using commercially available transfection reagent and cultured in 30 mL of FreeStyle™ 293 Expression Medium. Emission intensity of cell supernatants was measured by Luciferase assay, 96 hours post-transfection.

Expi293™, Freestyle™, Expi293F™ are registered trademarks of Thermo Fisher Scientific.

Transfection protocol for 30 ml culture volume

①		One day prior to transfection, prepare culture cells to be appropriate cell numbers for transfection using a 125mL or 250mL of Erlenmeyer shake flask . Note: Recommended seeding cell density is >0.8x106 viable cells/mL.
②		[Prepare cell suspension] Just before transfection, prepare 28mL of cell suspension with the cell densities below in a 125 mL of Erlenmeyer shake flask. ●For Expi293F™ Cells 2.7x106 viable cells/mL medium (7.5x107 viable cells/28mL of medium) ●For FreeStyle™ 293-F Cells 1.1x106 viable cells/mL medium (3.0x107 viable cells/28mL of medium) Note: cell viability must not be < 90 %.
③		Mix ScreenFect™UP-293 Transfection Reagent well with a vortex mixer.
④		[Prepare a diluted transfection reagent] ●For Expi293F™ Cells Add 60µL of ScreenFect™UP-293 Transfection Reagent into 940µL of ScreenFect™UP-293 Dilution Buffer in a sterile microfuge tube and mix it using pipette strokes. ●For FreeStyle™ 293-F Cells Add 30µL of ScreenFect™UP-293 Transfection Reagent into 970µL of Opti-MEM™ I Reduced Serum Medium in a sterile microfuge tube and mix it using pipette strokes.
⑤		[Prepare a diluted DNA solution] ●For Expi293F™ Cells Add 30µg of plasmid DNA with a density of 1µg/µL into 970µL of ScreenFect™UP-293 Dilution Buffer in a sterile microfuge tube and mix it using pipette strokes. ●For FreeStyle™ 293-F Cells Add 30µg of plasmid DNA with a density of 1µg/µL into 970µL of Opti-MEM™ I Reduced Serum Medium in a sterile microfuge tube and mix it using pipette strokes.
⑥		[Prepare a lipoplex solution] Mix the diluted transfection reagent and the diluted DNA solution with rapid pipette strokes and spin down it. Incubate it for 5-20minutes at room temperature. Note: Do not vortex the mixed solution.
⑦		Add the 2mL of the lipoplex solution into the cell suspension in the Erlenmeyer Shaker Flask with slowly swirling. Note: The final volume should be approximately 30mL in the flask.
⑧		Incubate the cells at 37°C(8% CO2), rotating at 125rpm on an orbital shaker for 16hours.
⑨		Add 150µL of ScreenFect™UP-293 Booster to the cells.
⑩		Continue to incubate the cells at 37°C(8% CO2), rotating at 125rpm on an orbital shaker. Collect cells or culture supernatants containing target proteins in 48-120 hours after transfection. Note: The incubation period required for successful transfection depends on the target protein and cell viability.

Expi293F™, Freestyle™ and Opti-MEM™ are registered trademarks of Thermo Fisher Scientific.

Kit Contents

＊ScreenFect™UP-293 Dilution Buffer is not necessary for the use of FreeStyle™ 293 Expression System.

Materials required

＊Use ScreenFect™UP-293 Dilution Buffer provided with this kit.

•  ∙∙∙ Store at 2 to 10°C
 
•  ∙∙∙ Store at −20°C
 
•  ∙∙∙ Store at −80°C
 
• Store at room temperature if not specified.
•  ∙∙∙ Specified poisonous substance
 
•  ∙∙∙ Poisonous substance
 
•  ∙∙∙ Deleterious substance
•  ∙∙∙ Class 1 Specified Chemical Substance by the Evaluation of Chemical Substances and Regulation of Their Manufacture, etc. (CSCL)
•  ∙∙∙ Class 2 Specified Chemical Substance by the CSCL
 
•  ∙∙∙ Poisonous substance
 
•  ∙∙∙ Deleterious substance
•  ∙∙∙ Class 1 designated substance by the Act on the Prohibition of Chemical Weapons and the Regulation of Specific Chemicals,
•  ∙∙∙ Class 2 designated substance by the same act
 
•  ∙∙∙ Psychotropic
 
•  ∙∙∙ Raw material of specified narcotics and psychotropics
•  ∙∙∙ Cartagena Protocol

* The details given show the information available as of April 2019.

The reagent given is intended only for the purposes of testing and research and cannot be used as a “drug”, “food” or “household product”.
Suggested delivery prices show the “price of the product alone” not including consumption tax.
Suggested delivery prices are the prices at the time of preparation of this article.

ScreenFect™ References

1. Diefenbacher, Markus E., et al. "The LIM Domain Protein nTRIP6 Recruits the Mediator Complex to AP-1-Regulated Promoters." PLoS ONE 9.5 (2014): e97549.
2. Freise, Christian, and Uwe Querfeld. "Inhibition of vascular calcification by block of intermediate conductance calcium-activated potassium channels with TRAM-34." Pharmacological Research (2014).
3. Hagiwara, Akane, et al. "Luteinizing Hormone-Induced Expression of Ptger4b, a Prostaglandin E2 Receptor Indispensable for Ovulation of the Medaka Oryzias latipes, Is Regulated by a Genomic Mechanism Involving Nuclear Progestin Receptor."
4. Peng, Yanyan, Ruidan Xu, and Xiaofeng Zheng. "HSCARG Negatively Regulates the Cellular Antiviral RIG-I Like Receptor Signaling Pathway by Inhibiting TRAF3 Ubiquitination via Recruiting OTUB1." PLoS pathogens 10.4 (2014): e1004041. (3)
5. Wakimoto, Hiroaki, et al. "Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas." Clinical Cancer Research (2014). (2)
6. Fischer, Simon, et al. "Breaking limitations of complex culture media: Functional non-viral miRNA delivery into pharmaceutical production cell lines." Journal of biotechnology 168.4 (2013): 589-600.
7. Bai, Dongmei, et al. "Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress." Nucleic acids research 42.3 (2014): 1799-1811.
8. Liu, Xing, et al. "Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi‐specific monoclonal antibody MsMab‐1." Cancer medicine 2.6 (2013): 803-814.