Cellfectin 2 reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, China

Cellfectin II reagent is a cationic lipid-based transfection reagent designed for efficient delivery of nucleic acids into a variety of mammalian cell types. It is used for the introduction of DNA, RNA, or other macromolecules into cells in culture.

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Cellfectin II (97 citations) Cellfectin (66 citations) Cellfectin reagent (33 citations) Cellfectin II transfection reagent (21 citations)

192 protocols using cellfectin 2 reagent

Rescue and Amplification of Recombinant Baculovirus for RVFV Protein Expression

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Rescue of the recombinant baculovirus, rpFB1-Gn head-PA, was done by transfection of Sf9 cells with the recombinant bacmid, pFBac 1-HBM-Gn head-PA, using the Cellfectin II Reagent (Life Technologies, United States). Briefly, Sf9 cells were seeded 24 h prior to transfection in a six-well plate at a density of 8 × 10⁵ cells/well to achieve a confluency of 80–90%. For transfection, 3 μg of recombinant bacmid was diluted in 100 μL Grace‘s Insect Medium (Thermo Fisher Scientific, United States) and combined with 8 μL Cellfectin II Reagent diluted in Grace‘s Insect Medium to a final volume of 210 μL. The resulting transfection mixture was vortexed briefly and incubated at room temperature for 15–30 min, after which the DNA–lipid mixture was added onto the Sf9 cells and incubated at 27°C. Supernatants containing first generation (P1) recombinant baculovirus were harvested 72 h post-transfection and passaged for three additional generations on Sf9 cells to yield fourth generation (P4) recombinant baculovirus. All passaging was done upon observation of cytopathic effects (CPE), typically within 24 h post-infection. Expression of the RVFV Gn head-PA fusion gene was confirmed by PCR (primers in Table 1) for both P3 and P4 recombinant baculovirus.

Zhang S., Yan F., Liu D., Li E., Feng N., Xu S., Wang H., Gao Y., Yang S., Zhao Y, & Xia X. (2022). Bacterium-Like Particles Displaying the Rift Valley Fever Virus Gn Head Protein Induces Efficacious Immune Responses in Immunized Mice. Frontiers in Microbiology, 13, 799942.

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Baculovirus Production in Insect Cells

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Baculoviruses were produced in Sf21 insect cells (EMBL Protein Expression and Purification Core Facility, Heidelberg Germany) using cellfectin II reagent (Thermo Fisher Scientific). The second virus generation was amplified in 50 ml (1 × 10⁶ cells/ml) culture. Afterwards, baculoviruses were harvested and diluted 1:100 in 100−400 ml (1−2 × 10⁶ cells/ml) expression culture in Sf21 or High Five cell line (BTI-TN-5B1-4, cat no. B855-02, Invitrogen) using Sf-900 III medium (Thermo Fisher Scientific) supplemented with 100 units/ml penicillin/100 µg/ml streptomycin (Thermo Fisher Scientific). Expression was done shaking at 27 °C for 60 h. Cells were harvested via centrifugation (800×g for 5 min), flash frozen in liquid N₂ and stored at −80 °C until further usage.

Zupa E., Würtz M., Neuner A., Hoffmann T., Rettel M., Böhler A., Vermeulen B.J., Eustermann S., Schiebel E, & Pfeffer S. (2022). The augmin complex architecture reveals structural insights into microtubule branching. Nature Communications, 13, 5635.

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Transfection of miRNA Mimics in Sf21 Cells

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mirVana^TM miRNA mimics were ordered for SpltNPV miRNAs 11684_3p and 11698_3p (ThermoFisher Scientific). Both the mimics were transfected in 70–80% confluent Sf21 cells at three concentrations i.e 10 µM, 50 µM and 100 µM using recommended concentration of Cellfectin II reagent (ThermoFisher Scientific). The transfection was carried out at room temperature for four hours in serum-free TNM-FH medium (Sigma-Aldrich) with intermittent shaking. Scrambled miRNA mimic was also transfected at a concentration of 10 µM, 50 µM and 100 µM in a similar fashion. After four hours, the medium was replaced with 10% FBS containing TNM-FH medium and the cells were maintained for 48 h.

Nayyar N., Kaur I., Malhotra P, & Bhatnagar R.K. (2017). Quantitative proteomics of Sf21 cells during Baculovirus infection reveals progressive host proteome changes and its regulation by viral miRNA. Scientific Reports, 7, 10902.

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Recombinant Bacmid Generation for ATP13A2 Expression

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Recombinant bacmids were generated from the pFastBac1 constructs using the Bac-to-Bac baculovirus expression system. DH10Bac E. coli competent cells (Thermo Fisher Scientific) were transformed with each pFastBac1-ATP13A2-GFP construct, and colonies were selected on an LB agar plate containing 50 μg/mL kanamycin, 7 μg/mL gentamycin, 10 μg/mL tetracycline, 100 μg/mL Bluo-Gal, 40 μg/mL IPTG. Bacmids were isolated and used for transfection of Sf9 cells. Sf9 cells were cultured in ESF 921 medium (Expression Systems) at 27 °C.
Baculovirus were generated by transfecting Sf9 cells using Cellfectin II reagent (Thermo Fisher Scientific), according to the standard Bac-to-Bac protocol or linear polyethylenimidine (PEI MAX; Polysciences) (Scholz and Suppmann, 2017 (link)). When necessary, baculovirus was amplified by infecting Sf9 cells at a 1:1000 volume-to-volume ratio and harvesting the culture supernatant after 4 days post-infection. For protein expression, Sf9 cells were infected at ~1.5–2.0 × 10⁶ cells/mL. Expression of ATP13A2 was monitored by GFP fluorescence of infected cells. Cells were harvested by centrifugation (1,500g for 7 min), 2–3 days post-infection, before any substantial cell death. Cell pellets were frozen in liquid nitrogen and stored at −80 °C until use.

Sim S.I., von Bülow S., Hummer G, & Park E. (2021). Structural basis of polyamine transport by human ATP13A2 (PARK9). Molecular cell, 81(22), 4635-4649.e8.

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Expression and Purification of Human DPP4

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The extracellular domain of human DPP4 (residues 39–766) was expressed using a Bac-to-Bac baculovirus expression system. In brief, the DNA encoding hDPP4 was cloned into the EcoR I and Hind III sites of pFastBac1. The final construct contains the N-terminal melittin signal peptide to facilitate secretion and a C-terminal 6× histidine tag for purification. The constructed DNA was then transformed into bacterial DH10Bac competent cells (Weidi Biotechnology, catalog number: DE1070, Shanghai, China), and the recombined bacmid DNA was extracted and transfected into Sf9 cells using Cellfectin II Reagent (Thermo Fisher Scientific, catalog number: 10362100, Waltham, MA, USA). Recombinant baculovirus was generated by transposition using the Bac-to-Bac system. The virus was used to infect High Five cells. The secreted glycosylated recombinant protein was isolated from the cell culture supernatant containing the target protein and harvested 72 h after infection. The target protein was loaded onto nickel (Ni)-charged resin (GE Healthcare, catalog number: 17–5247, Little Chalfont, Bukinghamshire, UK) and eluted with 0.25 M imidazole.

Li T.T., Peng C., Wang J.Q., Xu Z.J., Su M.B., Li J., Zhu W.L, & Li J.Y. (2021). Distal mutation V486M disrupts the catalytic activity of DPP4 by affecting the flap of the propeller domain. Acta Pharmacologica Sinica, 43(8), 2147-2155.

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Baculovirus Expression of ER Proteins

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The baculovirus system was used to express BAP31, DnaJB11, DnaJB11C, P7-1N, P7-1C, or P7-1, as described previously [47 (link)]. Recombinant bacmids were transfected into Sf9 cells using the Cellfectin II Reagent (Thermo Fisher Scientific). Sf9 cells infected with recombinant bacmids were processed for immunofluorescence microscopy. Meanwhile, Sf9 cells infected with recombinant bacmids were stained with the ER-Tracker Dyes (Thermo Fisher Scientific) for 30 min, and then processed for immunofluorescence microscopy to observe the relationship between ER and BAP31, DnaJB11C, P7-1N, P7-1C, or P7-1.

Yu X., Jia D., Wang Z., Li G., Chen M., Liang Q., Zhou Y., Liu H., Xiao M., Li S., Chen Q., Chen H, & Wei T. (2021). A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions. PLoS Pathogens, 17(3), e1009347.

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Sf21 Cell Culture and Baculovirus Titration

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The Spodoptera frugiperda Sf21 cell line was cultured at 28 °C in TNM-FH culture media (PAN-Biotech GmbH, Aidenbach, Germany) supplemented with 10% heat-inactivated foetal bovine serum (PAN-Biotech GmbH, Germany), gentamicin at 50 µg/mL (Sigma, St. Louis, MO, USA (Merck)), and Antibiotic-Antimicotic 1× (Sigma, St. Louis, MO, USA (Merck)). Recombinant baculoviruses (rBacs) were generated using the TopBac expression cassette [36 (link),37 (link)] as donor plasmids to generate the bacmids with the Bac-To-Bac baculovirus expression system (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Bacmids were transfected into Sf21 cells using Cellfectin^® II Reagent (Thermo Fisher Scientific) and following the manufacturer’s instructions.
Baculovirus stocks were titered on Sf21 cells using the 6-well plate format. Subconfluent Sf21 cells were infected with 10-fold serial dilutions of recBac stocks for 1 h at 28 °C. Subsequently, inocula were removed and cells were overlayed with an insect cell media-agarose mix at 1% agarose final concentration (UltraPure™ Low Melting Point Agarose, Life Technologies, Carlsbad, CA, USA) and further incubated for 3–5 days. Monolayers were stained with neutral red to facilitate plaque counting.

Dalton K.P., Alvarado C., Reytor E., del Carmen Nuñez M., Podadera A., Martínez-Alonso D., Alonso J.M., Nicieza I., Gómez-Sebastián S., Dalton R.M., Parra F, & Escribano J.M. (2021). Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses. Vaccines, 9(9), 1005.

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Production of Recombinant Baculoviruses

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According to the instructions of the manufacturer, Sf9 cells were transfected with the recombinant shuttle plasmids of rBacmidVP2, rBacmidL₂B, rBacmidNT₁L₂B, rBacmidNT₁L₂B₄B, and rBacmidN(T₁)₂L₂B₄B with Cellfectin^® II Reagent (Thermo Fisher Scientific Inc., USA), respectively. A liposome transfection reagent, after cytopathogenic changes were observed, the supernatants were collected and the recombinant viruses isolated and designated as rBacVP2, rBacL₂B, rBacNT₁L₂B, rBacNT₁L₂B₄B, and rBacN(T₁)₂L₂B₄B, respectively.

Li Q., Ma X., Shen Y., Dai J., Nian X., Shang X., Chen J., Wubshet A.K., Zhang J, & Zheng H. (2024). Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice. Viruses, 16(4), 621.

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Drosophila S2 Cell Transfection and Recombinant Protein Purification

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Drosophila S2 cells were grown and passaged in Schneider’s Drosophila medium (ThermoFisher Scientific) plus 10% heat-inactivated fetal bovine serum (FBS). For transfections, 1 × 10⁶ cells/well were seeded in 1 mL complete medium in six-well plates. Before transfection, medium was removed and replaced with FBS-free Schneider’s Drosophila medium. pAMW plasmids were transfected with Cellfectin™ II Reagent (ThermoFisher) and Opti-MEM medium (ThermoFisher) according to manufacturer’s instructions with 6 μg plasmid/well. After 72 h, cells were collected by scraping and lysed in Buffer D + PI + DTT. Full-length open reading frame of JARID2 was cloned into pFastBac, using the Bac-to-Bac N-His TOPO cloning kit (Life Technologies) for baculovirus generation via Bac-to-Bac baculovirus expression from the St. Jude Protein Production Facility. Full-length recombinant protein was bound overnight at 4 °C to Pierce™ Protein A/G Magnetic Beads (ThermoFisher Scientific) with JARID2 antibody (Novus Biologicals). Protein-bead complexes (or beads with no antibody) were washed with PBST, added to recombinant protein fragment lysate for 4 h at 4 °C in HEPM, washed in PBST, and eluted with 0.1 M glycine (pH 2.3). Eluates were neutralized with 1.5 M Tris buffer, pH 8.8, and analyzed by Western blotting.

Evans M.K., Matsui Y., Xu B., Willis C., Loome J., Milburn L., Fan Y., Pagala V, & Peng J.C. (2020). Ybx1 fine-tunes PRC2 activities to control embryonic brain development. Nature Communications, 11, 4060.

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Baculovirus-Mediated AAV Production Protocol

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Inducible expression plasmids (pCLDs) and Nano-Luc reporter constructs were created using GeneArt services (Thermo Fisher Scientific, Waltham, MA). To generate a recombinant baculovirus containing only the ITR-transgene-ITR (Bac Trans), the AAV Cap expression cassette alone (Bac polH Cap2/5),⁸ or the AAV Cap expression cassette and ITR-transgene-ITR (Bac polH Cap5 [human Factor IX (FIX)] or Bac polH Cap2/5 [secreted Nano-Luc (sNano-Luc)]), Sf9 cells were transfected with pVD-ITR-transgene-ITR (SEAP transgene) or pVD-polH-Cap (polH Cap2) or pVD-polH-Cap-ITR-transgene-ITR (polH Cap Trans) (Cap5 FIX, Cap2/5 sNano-Luc) and linearized baculovirus genome using Cellfectin II reagent (Thermo Fisher Scientific, Waltham, MA). The in-house-developed linear baculovirus genome contains the lacZ gene, which is replaced after successful homologous recombination with the plasmid DNA. Positive cell plaques were identified using blue-white screening and transferred onto adherent Sf9 cells. 72 h after transfection, the supernatant from Sf9 cells was passaged and amplified in ExpresSf+ cells until passage 4 and stored in liquid nitrogen. The baculovirus expressing AAV2 Rep (Bac Rep183) was generated as described previously.⁸ Bac Rep183 is also called the double cassette AAV Rep.

Moreno F., Lip F., Rojas H, & A.n.g.g.a.k.u.s.u.m.a. (2022). Development of an insect cell-based adeno-associated virus packaging cell line employing advanced Rep gene expression control system. Molecular Therapy. Methods & Clinical Development, 27, 391-403.

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