Fugene 6 transfection reagent

Manufactured by Promega

Sourced in United States, United Kingdom, Japan, France, China, Sweden

FuGENE 6 is a non-liposomal transfection reagent used to deliver nucleic acids into eukaryotic cells. It facilitates the uptake of DNA, RNA, or proteins into cells through a proprietary formulation.

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Lab products found in correlation

711 protocols using fugene 6 transfection reagent

Overexpression and Knockdown Protocols for DDR1 and MT1-MMP in MDA-MB-231 Cells

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DDR1-GFP overexpression was performed with pLVX-CMV-DDR1-GFP. DDR1-GFP lentiviral particles were generated through co-transfection of 293 T cells with pCMV ΔR8.91 (gag-pol) and phCMVG-VSVG (env) expression constructs using the FuGene 6 transfection reagent (Promega) according to the manufacturer’s recommendations. Three days after transfection, the viral supernatant mixed with fresh medium (1 of 4) and hexadimethrine bromide at 8μg/ml (Sigma) was used to infect MDA-MB-231 cells. Infected cells were selected using puromycin (Invivogen) at 1 μg/ml.
MT1-MMP knock-down was achieved with MMP-14-specific shRNAs (Buache et al., 2014 (link)). shRNA MMP-14 retroviral particles were generated through co-transfection of 293 T cells with pCL-Ampho expression constructs using the FuGene 6 transfection reagent (Promega) according to the manufacturer’s recommendations. Three days after transfection, the viral supernatant mixed with fresh medium (1 of 4) and hexadimethrine bromide at 8μg/ml (Sigma) was used to infect MDA-MB-231 cells. Infected cells were selected using puromycin (Invivogen) at 1 μg/ml.

Saby C., Collin G., Sinane M., Buache E., Van Gulick L., Saltel F., Maquoi E, & Morjani H. (2019). DDR1 and MT1-MMP Expression Levels Are Determinant for Triggering BIK-Mediated Apoptosis by 3D Type I Collagen Matrix in Invasive Basal-Like Breast Carcinoma Cells. Frontiers in Pharmacology, 10, 462.

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Lentiviral Production and Transduction Protocol

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For lentivirus production, 3 million HEK293T cells were plated on a T-75 flask. One day after plating, cells were transfected with a prepared mix of DMEM containing 5 μg of base editor construct or lentiGuide-SLC10A-Puro with 3.75 μg of psPAX2 (Addgene #12260) and 2.5 μg of pMD2.G (Addgene #12259) using FuGENE 6 transfection reagent (Promega, Madison, WI). 24 h after transfection, the medium was replaced with DMEM with 10% BSA, and supernatants were harvested every day up to 3 days after transfection. Supernatants containing lentivirus were concentrated by ultracentrifugation (25,000 rpm, 4°C, 90 min). The concentrated lentivirus was resuspended in 200 μL of PBS and stored at −80°C until infection.
For transduction, cells were plated on 6-well plates on the day before transduction and transduced with concentrated lentivirus. Two days after transduction, cells were selected in G418 (0.5–0.75 mg/mL) or puromycin (1.5 μg/mL for HepG2-NTCP and 5 μg/mL for stem cells). For the transfection into stem cells, cells were transfected with 2 μg of base editor construct and 1.5 μg of lentiGuide-SLC10A-Puro on 6-well plates using FuGENE 6 transfection reagent (Promega).

Uchida T., Park S.B., Inuzuka T., Zhang M., Allen J.N., Chayama K, & Liang T.J. (2021). Genetically edited hepatic cells expressing the NTCP-S267F variant are resistant to hepatitis B virus infection. Molecular Therapy. Methods & Clinical Development, 23, 597-605.

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CRISPR-Cas9 Knockouts of Key Signaling Proteins

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RIG-I, STING, STAT2, and p65 CRISPR-Cas9 KO cell lines were performed by transfection with Lipofectamine CRISPRMAX of TrueGuide Synthetic CRISPR gRNA and TrueCut Cas9 Protein v2 according to the manufacturer’s instructions (Thermo Fisher Scientific). CRISPR gene-editing efficiency was verified using GeneArt Genomic Cleavage Detection kit (A24372; Thermo Fisher Scientific). MAVS KO cell lines were performed by transfection of the pSpCas9(BB)-2A-Puro (PX459) plasmid containing MAVS gRNAs with FuGENE 6 Transfection Reagent (E2691; Promega). Sixteen hours after transfection, cells were selected with puromycin (1 μg/ml) for 2 days. IRF3 KO cell lines were performed by transfection of the pU6-(BbsI)-CBh-Cas9-T2A-mCherry plasmid containing IRF3 gRNAs with FuGENE 6 Transfection Reagent (E2691; Promega). Twenty-four hours after transfection, mCherry-expressing cells were single cell-sorted into 96-well plates. MCF10A IRF3 KO and MCF10A p53 KO cell lines was created as previously described^{66 ,84 (link)}. For every target, three or more independent clones were generated. gRNA sequences used in this study are listed in Supplementary Table 2.

Oh S., Bournique E., Bowen D., Jalili P., Sanchez A., Ward I., Dananberg A., Manjunath L., Tran G.P., Semler B.L., Maciejowski J., Seldin M, & Buisson R. (2021). Genotoxic stress and viral infection induce transient expression of APOBEC3A and pro-inflammatory genes through two distinct pathways. Nature Communications, 12, 4917.

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Overexpression of Murine Igsf3 in HEK293FT Cells

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Human embryonic kidney (HEK293FT) cells (ATCC) were cultured in DMEM high glucose (4.5 g/L) with L-glutamine (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (both from BioNordika). HEK293FT cells were plated one day before transfection and were approximately 60% confluent on the day of transfection. The murine Igsf3 plasmid (ORFeome Library) was transfected into HEK293FT cells by using the FuGENE6 transfection reagent (Promega) at a 3:1 ratio of FuGENE6 transfection reagent to DNA according to the manufacturer’s protocol. Cells were incubated for 48 h, lysed, and the extracts were analyzed for IGSF3 expression by Western blotting. Mock-transfected cells (plasmid without Igsf3) and WT HEK293FT cells were used as controls.

Tanjore Ramanathan J., Zárybnický T., Filppu P., Monzo H.J., Monni O., Tervonen T.A., Klefström J., Kerosuo L., Kuure S, & Laakkonen P. (2023). Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization. Scientific Reports, 13, 17162.

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HEK293 Cell Transfection of hClC-1 Mutants

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HEK293 cells (American Type Culture Collection, USA) were incubated in Dulbecco’s modified Eagle’s medium (Hyclone), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ug/ml streptomycin according to the supplier’s recommendations. hClC-1 and mutant channels were transiently expressed using fugene 6 transfection reagent (Promega), based on manufacturer’s guide. To mimic heterozygous effect of autosomal dominant pattern, mutants were co-expressed in 1:1 ratio with WT, using fugene 6 transfection reagent (Promega) and incubated in 37°C. All experiments were conducted after 24–48 h of transfection.

Ha K., Kim S.Y., Hong C., Myeong J., Shin J.H., Kim D.S., Jeon J.H, & So I. (2014). Electrophysiological Characteristics of Six Mutations in hClC-1 of Korean Patients with Myotonia Congenita. Molecules and Cells, 37(3), 202-212.

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Overexpression and Knockdown of DDR1 and LRP-1 in HT-29 Cells

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DDR1-GFP overexpression was performed using pLVX-CMV-DDR1-GFP construct which was a generous gift from Frederic Saltel (INSERM, UMR1053, BaRITOn Bordeaux Research in Translational Oncology, Bordeaux, France). DDR1-GFP lentiviral particles were generated by transient co-transfection of 293T with pCMV ΔR8.91 (gag-pol) and phCMVG-VSVG (env) expression constructs using the FuGene 6 transfection reagent (Promega) according to manufacturer’s recommendations. Three days after transfection, the supernatant containing lentiviruses was collected, filtered through 0.45 μm filter, mixed with fresh medium (1 of 4) and hexadimethrine bromide at 8 μg/ml (Sigma) and used to infect HT-29 recipient cells. GFP control cells were processed in the same way. Infected cells were selected using puromycin (Invitrogen) at 3 μg/ml. LRP-1 knock-down was achieved using shRNA sequences previously described (Dedieu et al., 2008 (link)) that were purchased from Sigma. shRNA LRP-1 lentiviral particles were produced in 293T cells using FuGene 6 transfection reagent (Promega) and used to infect HT-29 recipient cells as described above. HT-29 cells expressing control shRNA were generated in the same way. Infected cells were selected using puromycin (Invitrogen) at 3 μg/ml.

Le C.C., Bennasroune A., Collin G., Hachet C., Lehrter V., Rioult D., Dedieu S., Morjani H, & Appert-Collin A. (2020). LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis. Frontiers in Cell and Developmental Biology, 8, 412.

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Optimized Transfection of HEK293T Cells

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Transfection was optimized in HEK293T cells using Fugene 6 transfection reagent (Promega, Madison, WI, USA). HEK293T cells were cultured to a density of ~70% on the day of transfection. Our smacATPi and neg-smacATPi plasmids were transfected into HEK293T cells using Fugene6 transfection reagent (Promega, WI, USA) following the manufacturer’s instructions. At 24 h post-transfection, cells were imaged during drug trial runs and analysis. The transfection efficiency of the smacATPi indicator for HEK293T cells was ~80–90%.

White D I.I.I., Lauterboeck L., Mobasheran P., Kitaguchi T., Chaanine A.H, & Yang Q. (2023). Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells. Cells, 12(5), 695.

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Purification and Deacylation of Recombinant SIRT6

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Full-length human SIRT6 WT or mutant was inserted into pCMV-Tag 4a vector with C-terminal Flag tag. The SIRT6 WT and different mutant plasmids were transfected into HEK 293T cells using FuGene 6 transfection reagent according to the manufacturer’s protocol. pCMV-Tag 4a empty vector was used as negative control. The cells were collected at 500 g for 5 min and then lysed in Nonidet P-40 lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol and 1 % Nonidet P-40) with universal nuclease (1:1000 dilution) and protease inhibitor cocktail (1:100 dilution) at 4 °C for 30 min. After centrifuging at 15,000 g for 15 min, the supernatant was collected and incubated with 20 µL of anti-Flag affinity gel at 4 °C for 2 h. The affinity gel was washed three times with washing buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2% Nonidet P-40) and then eluted by 3X FLAG peptide (dissolved in 25 mM Tris-HCl pH 7.4, 150 mM NaCl and 10% glycerol). Eluted SIRT6 proteins were buffer exchanged three times using Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane. SIRT6 proteins after buffer exchange were used for the deacylation assay.

Zhang X., Khan S., Jiang H., Antonyak M.A., Chen X., Spiegelman N.A., Shrimp J.H., Cerione R.A, & Lin H. (2016). Identifying the functional contribution of the defatty-acylase activity of SIRT6. Nature chemical biology, 12(8), 614-620.

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Purification and Deacylation of Recombinant SIRT6

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Transfection of HEK293 cells with TDiP aggregates

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HEK293 cells were seeded to 8-chamber dishes (Cellvis #C8-1.5H-N) such that they reached 50% confluence 16 hours post-seeding. Cells were transfected with a control mCherry expression vector made in-house (pcDNA3.1-P2A-mCherry-T2A) using Fugene 6 Transfection reagent according to manufacturer protocol. 24 hours later, 10.0 μg (A₂₈₀) of TDiP aggregates per well (∼80 mm²), were transfected into cells using ProteoJuice Transfection reagent (Millipore Sigma #71281). Ratios were adapted for chamber dish from manufacturer protocol and De Rossi et al., 2021. 4 hours following transfection, cells were returned to complete media and imaged on a 37°C heated stage in a humidified, 5% CO₂ chamber.

Evangelista B.A., Cahalan S.R., Ragusa J.V., Mordant A., Necarsulmer J.C., Perna R.J., Ajit T., White K., Barker N.K., Tian X., Cohen S., Meeker R., Herring L.E, & Cohen T.J. (2023). Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates. iScience, 26(5), 106645.

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