Expi293f cells

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, United Kingdom

Expi293F cells are a suspension-adapted mammalian cell line derived from HEK293F cells. They are designed for high-level recombinant protein expression in biopharmaceutical and biotechnology applications.

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615 protocols using expi293f cells

Cell Line Authentication for Protein Production

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Cell line sources are as follows: HeLa cells for single-molecule imaging were from the German Collection of Microorganisms and Cell Cultures GmbH (ACC 57); Expi293F cells (Thermo Fisher); Expi293F GnTI- (Thermo Fisher); IL-17C HEK-Blue NF-kB Reporter cells (Invivogen). Cell lines were authenticated as follows: HeLa cells (German Collection of Microorganisms and Cell Cultures GmbH), Expi293F cells (Thermo Fisher), Expi293F GnTI⁻ (Thermo Fisher) and IL-17C HEK-Blue NF-κB reporter cells (Invivogen) were guaranteed by the suppliers and no additional authentication was performed by the authors of this study. HeLa cells for single-molecule imaging tested negatively for mycoplasma (PCR). Expi293F cells (Thermo Fisher) and Expi293F GnTI⁻ cells (Thermo Fisher) used for protein production were not tested for mycoplasma contamination by the authors of this study. The IL-17C HEK-Blue NF-κB reporter cells (Invivogen) were tested for mycoplasma contamination by the manufacturer and no additional testing was performed by the authors of this study.

Wilson S.C., Caveney N.A., Yen M., Pollmann C., Xiang X., Jude K.M., Hafer M., Tsutsumi N., Piehler J, & Garcia K.C. (2022). Organizing structural principles of the IL-17 ligand–receptor axis. Nature, 609(7927), 622-629.

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Expression and Purification of Neurological Proteins

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RTN4Rs, BAI ECDs, BAI TSR domain(s), four splice variants of Neuroligin 1 ECD used in SPR, C1ql3 and Teneurin2 ECD were cloned into pEG BacMam vectors and expressed in Expi293F™ cells (Thermo Fisher Scientific) following manufacturer’s protocol. Briefly, 50 mL of Expi293F™ cells were grown to 3 × 10⁶ cells/mL. 50 μg of DNA was transiently transfected to Expi293F™ cells using 135 μL GibcoTM ExpiFectamine™ 293 Reagent. Enhancers were added 18 hours after transfection and cells were incubated for another 72 hours before harvesting. Proteins were purified by Ni²⁺-NTA affinity column chromatography followed by SEC using 1 × HBS (10 mM HEPES, pH 7.2, 150 mM NaCl) buffer. For the site-specifically protein biotinylation, BirA biotin-protein ligase standard reaction kit (Avidity) was used. Successful biotinylation was confirmed by streptavidin shift assay. Basically, streptavidin (SA) (Thermo Fisher Scientific) was mixed with biotinylated protein, followed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) comparison of SA-protein complex with biotinylated protein alone. Similarly, BAI1 and RTN4R protein mutant constructs were made using site-directed mutagenesis to the original WT construct. The mutant proteins were expressed and purified as described for the WT protein.

Wang J., Miao Y., Wicklein R., Sun Z., Wang J., Jude K.M., Fernandes R.A., Merrill S.A., Wernig M., Garcia K.C, & Südhof T.C. (2021). RTN4/NoGo-Receptor Binding to BAI Adhesion-GPCRs Regulates Neuronal Development. Cell, 184(24), 5869-5885.e25.

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Transfection of HEK293 Cells for GFRAL and RET

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HEK293 cells (CRL‐1573) were obtained from the American Type Culture Collection (Manassas, VA, USA), Expi293F cells were obtained from Thermo Fisher Scientific, and B16F10‐luc‐G5 cells were obtained from Caliper Life Sciences (Hopkinton, MA, USA). The cells were cultured at 37°C with 5% CO₂. HEK293 and B16F10‐luc‐G5 cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum and 1X Antibiotic‐Antimycotic solution (15240062, Gibco; Thermo Fisher Scientific). Expi293F cells were cultured in Expi293 Expression Medium (A1435101, Gibco; Thermo Fisher Scientific). For in vitro analyses, HEK293 cells were transfected with vectors expressing human GFRAL (OHu31183D; GenScript, Piscataway, NJ, USA), human RET (HG11997‐CF; Sino Biological), and luciferase under a serum response element (SRE) promoter (SRE‐luciferase) (E1340; Promega, Madison, WI, USA) using Lipofectamine 3000 Transfection Reagent (L3000001; Thermo Fisher Scientific).

Lee B.Y., Jeong J., Jung I., Cho H., Jung D., Shin J., Park J., Park E., Noh S., Shin S., Kang S., Heo J., Baek M, & Yea K. (2023). GDNF family receptor alpha‐like antagonist antibody alleviates chemotherapy‐induced cachexia in melanoma‐bearing mice. Journal of Cachexia, Sarcopenia and Muscle, 14(3), 1441-1453.

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Recombinant Monoclonal Antibody Production

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Selected pairs of heavy and light chain sequences were synthesized by GenScript and sequentially cloned into IgG1, Igκ/γ and Fab expression vectors. Heavy and light chain plasmids were co-transfected into Expi293F cells (Thermo Fisher Scientific) for recombinant mAb production, followed by purification with protein A agarose resin (GoldBio). Expi293F cells were cultured in Expi293 Expression Medium (Gibco) according to the manufacturer’s protocol.

kim W., Zhou J.Q., Horvath S.C., Schmitz A.J., Sturtz A.J., Lei T., Liu Z., Kalaidina E., Thapa M., Alsoussi W.B., Haile A., Klebert M.K., Suessen T., Parra-Rodriguez L., Mudd P.A., Whelan S.P., Middleton W.D., Teefey S.A., Pusic I., O’Halloran J.A., Presti R.M., Turner J.S, & Ellebedy A.H. (2022). Germinal centre-driven maturation of B cell response to mRNA vaccination. Nature, 604(7904), 141-145.

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Maintaining FreeStyle and Expi293F Cells

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FreeStyle 293-F (cat# R79007) and Expi293F cells (cat# A14528; RRID: CVCL_D615) were purchased from ThermoFisher Scientific Inc. FreeStyle 293-F cells were maintained in FreeStyle 293 Expression Medium, while Expi293F cells were maintained in Expi Expression Medium. The above cell lines were used directly from the commercial sources and cultured according to manufacturer suggestions.

Shi W., Wang L., Zhou T., Sastry M., Yang E.S., Zhang Y., Chen M., Chen X., Choe M., Creanga A., Leung K., Olia A.S., Pegu A., Rawi R., Schön A., Shen C.H., Stancofski E.S., Talana C.A., Teng I.T., Wang S., Corbett K.S., Tsybovsky Y., Mascola J.R, & Kwong P.D. (2022). Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability. Structure (London, England : 1993), 30(9), 1233-1244.e7.

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Cell Culture Protocols for Diverse Cell Lines

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FreeStyle 293-F (cat# R79007), Expi293F cells (cat# A14635) were from Thermo Fisher Scientific. HEK293S GnTI- (cat# CRL-3022), HEK293T/17 (cat# CRL-11268), I1 mouse hybridoma (cat# CRL-2700) and Vero E6 cells (cat# CRL-1586) were from ATCC.
FreeStyle 293-F cells and were cultured in serum-free FreeStyle 293 Expression Medium (GIBCO, cat# 12338026) at 37°C, 10% CO₂, 115 rpm. HEK293S GnTI- cells were cultured in FreeStyle 293 Expression Medium at 37°C, 10% CO₂, 115 rpm. Expi293F cells were cultured in Expi293 Expression Medium (GIBCO, cat# A14635) at 37°C, 8% CO₂, 125 rpm. HEK293T/17 cells and Vero E6 cells were cultured in 10% Fetal Bovine Serum (FBS, GIBCO cat# 16140071) supplemented Dulbecco’s Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37°C, 5% CO₂. Cell lines were not specifically authenticated.

Cerutti G., Guo Y., Zhou T., Gorman J., Lee M., Rapp M., Reddem E.R., Yu J., Bahna F., Bimela J., Huang Y., Katsamba P.S., Liu L., Nair M.S., Rawi R., Olia A.S., Wang P., Zhang B., Chuang G.Y., Ho D.D., Sheng Z., Kwong P.D, & Shapiro L. (2021). Potent SARS-CoV-2 neutralizing antibodies directed against spike N-terminal domain target a single supersite. Cell Host & Microbe, 29(5), 819-833.e7.

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Sialylation of Recombinant Proteins

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Expi293F cells and the mammalian expression vector pcDNA3.1 were obtained from Thermo Fisher Scientific, USA. Cells were cultured in Expi293 expression medium (Thermo Fisher Scientific, USA). All tissue culture media were supplemented with antibiotic–antimycotic solution (Thermo Fisher Scientific, USA), and cells were maintained at 37 °C in incubators with an atmosphere of 8% CO₂. Transient transfections of expression plasmids using Expi293F cells were performed using Expi293 expression system according to the manufacturer's recommendations (Thermo Fisher Scientific, USA). All proteins were generated as highly sialylated entities via cotransfection of HuCR1-encoding plasmids with plasmids encoding B4GALT1 and ST3GAL3 at a DNA ratio of 94:3:3, respectively. Poorly sialylated recombinant protein was generated as previously, but without cotransfection of B4GALT1/ST3GAL3.

Wymann S., Dai Y., Nair A.G., Cao H., Powers G.A., Schnell A., Martin-Roussety G., Leong D., Simmonds J., Lieu K.G., de Souza M.J., Mischnik M., Taylor S., Ow S.Y., Spycher M., Butcher R.E., Pearse M., Zuercher A.W., Baz Morelli A., Panousis C., Wilson M.J., Rowe T, & Hardy M.P. (2020). A novel soluble complement receptor 1 fragment with enhanced therapeutic potential. The Journal of Biological Chemistry, 296, 100200.

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Cell Line Cultivation and Primary Bovine B-Cell Isolation

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The common laboratory cell lines HEK293T (ATCC CRL-11268), and Hela (ATCC CCL2) were originally purchased from ATCC. The source of other cell lines were: Expi293F cells (Thermo Fisher Scientific); and TZM-bl cells (AIDS Research and Reference Reagents Program, Division of AIDS ARP-8129). Master seed stocks of all cell lines were confirmed for identity through Short Tandem Repeat profiling and were negative for mycoplasma and were routinely tested bi-monthly. All cell lines were cultivated in complete Dulbecco’s modified Eagle’s medium (DMEM) media and 10% fetal bovine serum (FBS), except for Expi293F cells that were cultured in Expi293 Expression Medium (Gibco).
Primary bovine B-lymphocytes were prepared from bovine PBMCs obtained by centrifugation of cow blood on a Ficoll-Plaque PLUS step gradient (GE Healthcare).

Heydarchi B., Fong D.S., Gao H., Salazar-Quiroz N.A., Edwards J.M., Gonelli C.A., Grimley S., Aktepe T.E., Mackenzie C., Wales W.J., van Gils M.J., Cupo A., Rouiller I., Gooley P.R., Moore J.P., Sanders R.W., Montefiori D., Sethi A, & Purcell D.F. (2022). Broad and ultra-potent cross-clade neutralization of HIV-1 by a vaccine-induced CD4 binding site bovine antibody. Cell Reports Medicine, 3(5), 100635.

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Maintaining FreeStyle and Expi293F Cells

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Shi W., Wang L., Zhou T., Sastry M., Yang E.S., Zhang Y., Chen M., Chen X., Choe M., Creanga A., Leung K., Olia A.S., Pegu A., Rawi R., Shen C.H., Stancofski E.S., Talana C.A., Teng I.T., Wang S., Corbett K.S., Tsybovsky Y., Mascola J.R, & Kwong P.D. (2022). Vaccine-elicited murine antibody WS6 neutralizes diverse beta-coronaviruses by recognizing a helical stem supersite of vulnerability. bioRxiv.

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Norovirus Capsid Protein Expression

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E coli Top 10 with pVR21 plasmid containing norovirus capsid genes were propagated at 30°C in the presence of 0.1mg/ml carbenicillin in Luria-Bertani broth. For full IgG expression and characterization, VH and VL plasmids were transfected into 30 mL cultures of Expi293F cells (Invitrogen) at a 1:2 ratio and incubated at 37°C and 8% CO₂ for 7 days.
For protein crystallization, P-domains were transfected into E.coli BL21 (DE3) (Agilent Technologies) and plated onto Luria-Bertani plates with 50μg/mL Ampicillin (LB-Amp). VH and VL chains were cotransfected into Expi293F cells (ThermoFisher) using a 3:1 ratio of Turbo293 transfection reagent (Speed Biosystem) to plasmid. Cells were incubated at 37°C, 5% CO₂, 130 rpm shaking, 70% humidity for 18 h and subsequently boosted with 80mL of AbBooster (Abi Scientific).

Lindesmith L.C., McDaniel J.R., Changela A., Verardi R., Kerr S.A., Costantini V., Brewer-Jensen P.D., Mallory M.L., Voss W.N., Boutz D.R., Blazeck J.J., Ippolito G.C., Vinje J., Kwong P.D., Georgiou G, & Baric R.S. (2019). Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination. Immunity, 50(6), 1530-1541.e8.

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