Spodoptera frugiperda sf9 insect cells

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Spodoptera frugiperda (Sf9) insect cells are a common cell line used in research for the expression of recombinant proteins. These cells are derived from the ovarian tissue of the fall armyworm, Spodoptera frugiperda. Sf9 cells are widely used for the production of various proteins, including enzymes, antibodies, and viral proteins.

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8 protocols using spodoptera frugiperda sf9 insect cells

Human Y2 Receptor Expression and Purification

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To enable receptor expression and purification, the WT human Y₂R gene (Genewiz) was cloned into a modified pFastbac1 vector (Invitrogen) with a hemagglutinin signal sequence at the N terminus, and a PreScission protease site followed by a 10 × His-tag and a Flag tag at the C terminus. To improve protein yield and stability, two mutations H149^3.51Y and S280^6.47C were introduced into Y₂R and 28 amino acids (residues S354-V381) at the C terminus of Y₂R were truncated using standard QuikChange PCR. To facilitate crystallization, residues 2–161 of a modified T4L were fused to the receptor N terminus, and residues S251-N256 in ICL3 were replaced by residues 2–148 of a modified flavodoxin (P2A, Y98W)^{27 (link)} through overlap extension PCR. Sequences of all primers used in this work are shown in Supplementary Table 3.
High-titer recombinant baculovirus (>10⁸ viral particles per ml) of the modified Y₂R was prepared using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Spodoptera frugiperda (Sf9) insect cells (Invitrogen) were grown to a density of 2 × 10⁶ cells ml⁻¹ in ESF 921 serum-free medium (Expression Systems) at 27 °C and then infected with the viral stock at a multiplicity of infection of 5. The cell pellets were harvested by centrifugation 48 h post infection and stored at −80 °C until use.

Tang T., Hartig C., Chen Q., Zhao W., Kaiser A., Zhang X., Zhang H., Qu H., Yi C., Ma L., Han S., Zhao Q., Beck-Sickinger A.G, & Wu B. (2021). Structural basis for ligand recognition of the neuropeptide Y Y2 receptor. Nature Communications, 12, 737.

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Expression of 5-HT Receptor Complexes

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Human 5-HT_1A, 5-HT_1D, and 5-HT_1e, human DNGα_i1, human Gβ₁, and human Gγ₂ were co-expressed in Spodoptera frugiperda Sf9 insect cells (Invitrogen) using the baculovirus method (Expression Systems). For the 5-HT_1D–G_i and 5-HT_1e–G_i complexes, scFv16 was co-expressed to stabilize the protein. As for the apo–5-HT_1A–G_i (WT) complex used for GTPase assay, the DNGα_i1 was replaced by WTGα_i1. Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2-3 million cells per ml and then infected with four separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection and cell pellets were stored at -80°C.

Xu P., Huang S., Zhang H., Mao C., Zhou X.E., Cheng X., Simon I.A., Shen D.D., Yen H.Y., Robinson C.V., Harpsøe K., Svensson B., Guo J., Jiang H., Gloriam D.E., Melcher K., Jiang Y., Zhang Y, & Xu H.E. (2019). Structural insights into lipid and ligand regulation of serotonin receptors. Nature, 569(7755), 284-288.

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Recombinant FMDV Serotype A Protein Expression

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FMDV serotype A strain AF/72 was isolated and maintained in our laboratory (the strain was maintained and provided by the Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences (CAAS)). E. coli TOP10 competent cells, the baculovirus transfer vector pFastBac 1, and E. coli DH10Bac competent cells were purchased from Invitrogen (California, USA). Spodoptera frugiperda (Sf9) insect cells (Invitrogen, USA) were cultured in Sf-900™ II SFM medium (Invitrogen, USA) containing 5% heat-inactivated fetal bovine serum (FBS; Gibco, USA) at 27°C in an incubator with 5% CO₂. Restriction enzymes were purchased from New England Biolabs (NEB); the transfection Cellfectin® II Reagent and Grace's Insect Medium were purchased from Invitrogen; the mouse anti-His tag monoclonal antibody and HRP-conjugated goat anti-mouse IgG were purchased from Abbkine (California, USA).

Liu X., Lv J., Fang Y., Zhou P., Lu Y., Pan L., Zhang Z., Ma J., Zhang Y, & Wang Y. (2017). Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins. BioMed Research International, 2017, 7658970.

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Recombinant Respiratory Syncytial Virus Production

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Spodoptera frugiperda (Sf9) insect cells (Invitrogen, Waltham, MA, USA) were cultured in suspension using spinner flasks and SF900II medium (Invitrogen, Carlsbad, CA, USA) at 27 °C for the generation of recombinant baculovirus (rBV) and VLPs. HEp-2 cells were grown in tissue culture flasks using Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), penicillin and streptomycin at 37 °C with 5% CO₂. For polyclonal anti-RSV antibody acquisition, mice were infected thrice with RSV A2 virus at 4-week intervals. One week after the final infection, sera were collected via retro-orbital plexus puncture. Monoclonal anti-RSV fusion antibody (131-2A) was purchased from Millipore and used in virus plaque assay and Western blot. The mouse monoclonal influenza A virus anti-M1 antibody was purchased from Abcam (Cambridge, UK) and used in Western blot. Horseradish peroxidase (HRP)-conjugated secondary mouse IgG antibodies were purchased from Southern Biotech (Birmingham, AL, USA). Fluorophore-conjugated antibodies were purchased from BD Bioscience (Franklin Lakes, NJ, USA) and used to perform flow cytometry. RSV A2 was originally kindly provided by Dr. Marty Moore of Emory University. RSV A2 and FI-RSV were prepared following the method previously described [8 (link),22 (link),29 (link),30 (link)].

Lee S.H., Chu K.B., Kim M.J., Mao J., Eom G.D., Yoon K.W., Ahmed M.A, & Quan F.S. (2023). Virus-like Particle Vaccine Expressing the Respiratory Syncytial Virus Pre-Fusion and G Proteins Confers Protection against RSV Challenge Infection. Pharmaceutics, 15(3), 782.

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Baculovirus Production and Titration

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Spodoptera frugiperda (Sf9) insect cells (Thermo Fisher Scientific; Waltham, MA) were maintained in suspension in serum-free ESF921 medium (Thermo Fisher Scientific) at 28 °C. Human retinal epithelial ARPE-19 cells (The American Type Culture Collection [ATCC]; Manassas, VA) were plated into 24-well plates and grown in DMEM/F12 (1:1) medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT). HepG2 (ATCC) cells were cultured in DMEM with 10% FBS and used for baculovirus infection. The live-attenuated viral vaccine (v-Oka strain from ATCC; AR-795, prepared in BSL 2+ facility of Beijing Wantai Biopharm according to the modality of a varicella vaccine) was stored at −80 °C and was titrated using a plaque assay before experimentation. The recombinant baculovirus was constructed using the BacMagic system (Merck & Co Inc, Kenilworth, NJ, USA) and the progeny virus was stored at 4 °C. gE-specific monoclonal antibody, 1B11 [30 (link)] and HRP-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. The BacPAK Baculovirus Rapid Titer Kit for determining the titer of the baculovirus stock was purchased from Takara (Beijing, China).

Xue W., Li T., Zhang S., Wang Y., Hong M., Cui L., Wang H., Zhang Y., Chen T., Zhu R., Chen Z., Zhou L., Zhang R., Cheng T., Zheng Q., Zhang J., Gu Y., Xia N, & Li S. (2022). Baculovirus Display of Varicella–Zoster Virus Glycoprotein E Induces Robust Humoral and Cellular Immune Responses in Mice. Viruses, 14(8), 1785.

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Engineered H. sapiens Dynein-2 Constructs

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A H. sapiens cytoplasmic dynein-2 motor domain construct containing an N-terminal SNAP_f tag^{19 (link)} was modified to make V1 and V2 constructs. For V1, residues 4196–4201 were changed to sequence WIALNL and residues 4202–4307 (inclusive) were deleted. For V2, residues 2160–2211 (inclusive) in AAA2 were removed, corresponding to exon skipping in V2. Variants were introduced using PCR with Q5 polymerase (New England Biolabs, Ipswich, MA, USA). Dynein-2 constructs were expressed in Spodoptera frugiperda (Sf9) insect cells (Thermo Fisher Scientific, Waltham, MA, USA) using a baculovirus system as previously described.^{19 (link)} Protein purifications were performed at 4 °C. Proteins of interest were eluted by resuspending the resin in TEV buffer, adding 20 μg TEV protease, and incubating the reaction overnight on a roller. TEV-cleaved proteins were separated from the resin using an empty column (Supplementary Methods).

Vig A., Poulter J.A., Ottaviani D., Tavares E., Toropova K., Tracewska A.M., Mollica A., Kang J., Kehelwathugoda O., Paton T., Maynes J.T., Wheway G., Arno G., Khan K.N., McKibbin M., Toomes C., Ali M., Di Scipio M., Li S., Ellingford J., Black G., Webster A., Rydzanicz M., Stawiński P., Płoski R., Vincent A., Cheetham M.E., Inglehearn C.F., Roberts A, & Heon E. (2020). DYNC2H1 hypomorphic or retina-predominant variants cause nonsyndromic retinal degeneration. Genetics in Medicine, 22(12), 2041-2051.

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Recombinant Eicosanoid Production

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ARA, EPA, and DHA were purchased from Nacalai Tesque (Kyoto, Japan). (±)8-HEPE (comprising equal amounts of 8S-HEPE and 8R-HEPE), 8S-HEPE, (±)8-HETE, 8S-HETE, 8R-HETE, rac-10-HDHA, PD1, gamma-linoleic acid (GLA), stearidonic acid (SDA) and docosapentaenoic acid (DPA) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The Spodoptera frugiperda (Sf9) insect cells, and pFastBac vector were purchased from Thermo Fisher Scientific (Franklin, MA, USA). The anti-6xHis antibody [HIS.H8] was purchased from Abcam Japan (Tokyo, Japan).

Yuki S., Uemura A., Hakozaki M., Yano A., Abe M., Misawa Y., Baba N, & Yamada H. (2020). Identification of a novel enzyme from E. pacifica that acts as an eicosapentaenoic 8R-LOX and docosahexaenoic 10R-LOX. Scientific Reports, 10, 20592.

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Cultivation of Vero-76 Cells and PEDV-CO Strain

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Vero-76 cells (ATCC CRL-1587) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂. The Spodoptera frugiperda Sf9 insect cells were purchased from Thermo Fisher Scientific and cultured in the serum-free SF900 II medium (Gibco) in suspension. The PEDV Colorado (PEDV-CO) strain (GenBank: KF272920.1) obtained from the South Dakota Animal Disease Research and Diagnostic Laboratory (ADRDL) was propagated in Vero cells.

Yu J., Sreenivasan C., Uprety T., Gao R., Huang C., Lee E.J., Lawson S., Nelson J., Christopher-Hennings J., Kaushik R.S., Nelson E., Diel D.G., Hause B.M., Li F, & Wang D. (2021). Piglet immunization with a spike subunit vaccine enhances disease by porcine epidemic diarrhea virus. NPJ Vaccines, 6, 22.

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