Fugene 6

Manufactured by Promega

Sourced in United States, United Kingdom, China, Japan, Germany, Australia

FuGENE 6 is a transfection reagent that facilitates the delivery of nucleic acids, such as DNA and RNA, into eukaryotic cells. It is a proprietary, non-liposomal formulation that forms complexes with nucleic acids, enabling their efficient uptake by cells.

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Lab products found in correlation

1 499 protocols using fugene 6

Fluorescent Protein Labeling of ESCRT Proteins

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Human HeLa ‘‘Kyoto’’ (HeLa K) cells were maintained in DMEM (GIBCO) supplemented with 10% FBS, 100U/ml penicillin, and 100 mg/ml streptomycin at 37 °C under 5% CO2. Stable cell lines expressing fluorescently labelled ALIX, CHMP4B or TSG101 were previously described [16 (link), 89 (link)]. For transient transfection cells were incubated with mixture of FuGene 6 (Promega) and the plasmid of interest, using a ratio of 3:1 of FuGene 6 to DNA and incubated for 24–48 h. In most cases, cells were transfected in MatTek 3.5 cm dishes using 6 µl of FuGene 6 and 2 µg of DNA. Before cells were used for further analysis the medium was exchanged. The pEGFP-ALIX [59 (link)], pEGFP-Rab35 and pmCherry-Rab35 [90 (link), 91 (link)] plasmids were a kind gift from Dr. Arnaud Echard, the plasmid expressing Rab11-RFP was kindly provided by Dr. Kay Schink and the plasmid expressing mCitrine-KIF5B was kindly provided by Dr. Eva M. Wenzel. The plasmid expressing ALIX-mCherry [89 (link)], has been described previously.

Pust S., Brech A., Wegner C.S., Stenmark H, & Haglund K. (2023). Vesicle-mediated transport of ALIX and ESCRT-III to the intercellular bridge during cytokinesis. Cellular and Molecular Life Sciences, 80(8), 235.

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Stable Cell Line Generation

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Cells were dissociated and plated at a density of 5 × 10⁵ cells/well of a 6-well plate the night before. The next day, cells were transfected using Fugene 6 (Promega) at a ratio of 3 μg DNA/9 μL Fugene 6. piggyBac vectors containing indicated transgenes were transfected together with a transposase plasmid (System Biosciences, CA). Twenty-four hours later, fresh medium containing antibiotic selection was added to cells. Cells were maintained in selection medium until establishment of stable lines.

Ng Y.H., Chanda S., Janas J.A., Yang N., Kokubu Y., Südhof T.C, & Wernig M. (2021). Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics. Stem Cell Reports, 16(7), 1763-1776.

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Transfection of Nups, LANS, and Kap1

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Nups and LANS constructs were transfected 48 hours before imaging, with Fugene6 (Promega, Madison, WI). Kap1-mCherry and DN-Ran were transfected 16 hours before imaging. Cells were transfected with 1µg DNA (for LANS, Nup133-mEGFP, and Kap1-mCherry constructs), 500 ng DNA (BFP-DNRan constructs, Nup93-mEGFP), or 250 ng DNA (Nup54-mEGFP) and 3 µL Fugene6 (Promega) in Opti-Mem I (Gibco) to a final volume of 100 µL according to the manufacturer's instructions.

Pulupa J., Prior H., Johnson D.S., & Simon S.M. (2020). Cargo modulates the conformation of the nuclear pore in living cells.

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Transient and Stable Transfection Assays

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HEK293 cells (ATCC) were transfected with MSCV-N-GFP, MSCV-N-6E6, MSCV-N-11E6, and MSCV-N-16E6 plasmids using Fugene 6 (Promega). Forty-eight hours post transfection, cells were lysed and used for co-immunoprecipitation assay. For stable transfection, HEK293 cells were transfected with the same amount of MSCV-N-GFP and MSCV-N-E6 constructs using Fugene 6 (Promega) and selected with puromycin for 2 weeks. Then single clones were then isolated and propagated for co-immunoprecipitation assays.

Wang J., Dupuis C., Tyring S.K, & Underbrink M.P. (2016). Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins. PLoS ONE, 11(2), e0149859.

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Transfection of COS-1 Cells with hANO5-V5

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COS-1 cells (ATCC, Manassas, VA, USA) were cultured at +38°C, 5% CO2, in DMEM supplemented with 10% FCS, 1% PenStrep and 1% Glutamax. For transfection experiments, COS-1 cells were seeded on 6-well plates and grown to 60-70% confluence overnight. The next day, the cells were transfected with hANO5wt-V5 using FuGene 6 (Promega, Madison, WI, USA), with DNA (µg):FuGene 6 (µl) ratio 1:3. Two days after transfection the cells were harvested for western blotting.
Untransfected COS-1 cells were used as negative control.

Vihola A., Luque H., Savarese M., Penttilä S., Lindfors M., Leturcq F., Eymard B., Tasca G., Brais B., Conte T., Charton K., Richard I, & Udd B. (2018). Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy. Neuropathology and applied neurobiology, 44(5).

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Lentiviral Transduction Protocol

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Lentiviruses were produced in HEK293FT cells using the pPAX2 and pMD2.G packaging plasmids, kindly provided by Michael Way (London). HEK293FT cells were transfected with the packaging and transfer plasmids using FuGENE6 (Promega, Madison, WI) (7 μg pPAX2, 3 μg pMD2.G, 10 μg transfer plasmid complexed with 51 μl FuGENE6 in a 10 cm dish). Fresh medium containing 10 mM HEPES was provided 6–8 h.p.t.. Viruses were harvested in the next two days, pooled, filtered through 0.45 µm filters, aliquoted and frozen at −80°C for long-term storage.
For transduction experiments, viruses were diluted 1:16 in growth medium. U2OS cells were incubated in diluted virus overnight at 37°C. Fresh medium (containing 1 µg/ml puromycin if applicable) was provided the next day. Cells under selection were maintained in the presence of antibiotic for at least four days before being expanded and used for subsequent experiments.

Helle S.C., Feng Q., Aebersold M.J., Hirt L., Grüter R.R., Vahid A., Sirianni A., Mostowy S., Snedeker J.G., Šarić A., Idema T., Zambelli T, & Kornmann B. (2017). Mechanical force induces mitochondrial fission. eLife, 6, e30292.

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Transient Transfection of HTT and U1 Constructs

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Wild-type and mHTT and U1 minigene constructs were designed at PTC and synthesised at GenScript^®. For U1 constructs 5 × 10⁵ HEK293 cells were transfected with 2 µg of plasmid deoxyribonucleic acid (DNA) or mock control in six-well plates, using 6 µl Fugene6^® (Promega) according to the manufacturer’s instructions; after incubating for 24 h (37 °C, 5% CO₂, 100% relative humidity), cells were treated with either 1 µM HTT-C1 or 0.5% DMSO control and incubated for 48 h. For HTT constructs, 5 × 10⁵ HEK293 cells were transfected with 50 ng of plasmid DNA in 24-well plates, using 6 µl Fugene6^® according to the manufacturer’s instructions. After incubating overnight (37 °C, 5% CO₂, 100% relative humidity), cells were treated with varying concentrations of compounds in a final concentration 0.05% DMSO and incubated for 24 h.

Bhattacharyya A., Trotta C.R., Narasimhan J., Wiedinger K.J., Li W., Effenberger K.A., Woll M.G., Jani M.B., Risher N., Yeh S., Cheng Y., Sydorenko N., Moon Y.C., Karp G.M., Weetall M., Dakka A., Gabbeta V., Naryshkin N.A., Graci J.D., Tripodi T J.r., Southwell A., Hayden M., Colacino J.M, & Peltz S.W. (2021). Small molecule splicing modifiers with systemic HTT-lowering activity. Nature Communications, 12, 7299.

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Beta-Lactamase Reporter Assay for NMDA Receptors

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For the beta‐lactamase (β‐lactamase, β‐lac) reporter assay, HEK293 cells in 96‐well plates were transfected with cDNA encoding WT β‐lac‐GluN1, β‐lac‐GluN1‐M641I, β‐lac‐GluN1‐M641I/GluN2A, or β‐lac‐GluN1‐M641I/GluN2B using Fugene6 (Promega, Madison, WI).²³ The background absorbance was measured by cells transfected with Fugene6 only. A negative control for surface β‐lac activity was determined in cells that were transfected with WT β‐lac‐GluN1 or β‐lac‐GluN1‐M641I only. Eight wells were transfected for each condition, and the surface and the total β‐lac levels were measured in four wells each. The transfected cells were washed with HBSS and 10 mmol/L HEPES after 24‐h transfection. The surface activity was measured by adding 100 µL of a 100 µmol/L nitrocefin (Millipore, Burlington, MA) solution in HBSS with HEPES. The total activity was determined by a 30‐min incubation in 50 µL H₂O (to lyse the cells) before adding 50 µL of 200 µmol/L nitrocefin. The absorbance was determined by a microplate reader (SpectraMax M2; Molecular Devices) at 468 nm once every min for 30 min (30°C). The rate of the absorbance increase was calculated from the slope of the linear regression.²³

Xu Y., Song R., Chen W., Strong K., Shrey D., Gedela S., Traynelis S.F., Zhang G, & Yuan H. (2021). Recurrent seizure‐related GRIN1 variant: Molecular mechanism and targeted therapy. Annals of Clinical and Translational Neurology, 8(7), 1480-1494.

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miR-4284 Regulation of CXCL5 Expression

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Cells were seeded in 6 well plates (250,000 cells/well) and transfected with the mirVana miR-4284 mimic (Ambion) or its negative control, at a final concentration of 50 nM, using the transfection reagent FuGENE6 (Promega), according to the manufacturer’s guidelines. Cells were harvested 42 hours post-transfection and RNA was isolated.
Luciferase reporter plasmid psiCheck2-CXCL5-3’UTR carrying the 3’UTR of CXCL5 was used. Sequential transfection was performed; first, the reporter plasmid (4 µg plasmid per 1×10⁶ cells) was transfected using FuGENE6 (Promega) and 24 hours later, cells were transfected with a final concentration of 50 nM miRNA mimics/inhibitors or the respective negative controls, provided by the manufacturer, as described above.

Koukos G., Polytarchou C., Kaplan J.L., Oikonomopoulos A., Ziring D., Hommes D.W., Wahed R., Kokkotou E., Pothoulakis C., Winter H.S, & Iliopoulos D. (2015). A MicroRNA Signature in Pediatric Ulcerative Colitis: Deregulation of the miR-4284/CXCL5 pathway in the Intestinal Epithelium. Inflammatory bowel diseases, 21(5), 996-1005.

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Transfection Optimization for HEK293 and hCNCCs

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HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen, 11668019) at a ratio of 2.8 ul lipofectamine per ug of DNA, diluted in Opti-MEM. Cells were transfected with 2.5 ug DNA per well of a 6-well plate or 15 ug DNA per 10-cm plate 1–2 days after seeding, when they reached 70–90% confluency. Media was replaced 4–6h after transfection, and then cells were harvested for Western blot or chromatin immunoprecipitation at 24 h after transfection.
hCNCCs were transfected with FuGENE 6 (Promega, E2691) immediately after passaging, using 1 ul of FuGENE 6 per 3 ug of DNA and 100 ng DNA diluted in 50 ul Opti-MEM per well of a 24-well plate.

Kim S., Morgunova E., Naqvi S., Bader M., Koska M., Popov A., Luong C., Pogson A., Claes P., Taipale J, & Wysocka J. (2023). DNA-guided transcription factor cooperativity shapes face and limb mesenchyme. bioRxiv.

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