Fugene hd

Manufactured by Promega

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FuGENE HD is a non-liposomal transfection reagent designed for efficient delivery of DNA, RNA, and other macromolecules into a variety of cell types. It offers high transfection efficiency and low cytotoxicity.

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2 156 protocols using fugene hd

BRET Assay for PLK Engagement

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HEK293 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (Seradigm) with incubation in a humidified 37 °C/5% CO₂ incubator. N- or C-terminal NLuc/PLK fusions were encoded in pFN31K expression vectors (Promega), including flexible Gly-Ser-Ser-Gly linkers between NLuc and ORFs corresponding to UniProt isoform 1 (PLK1: P53350-1, PLK2: Q9NYY3-1, and PLK3: Q9H4B4-1). Carrier DNA was encoded in pGEM-3Z vectors (Promega). For cellular BRET target engagement experiments, HEK293 cells were transfected with NLuc/PLK fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, NLuc/PLK fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:9 (mass/mass), after which FuGENE HD was added at a ratio of 1:3 (µg DNA: µL FuGENE HD). One part (vol) of FuGENE HD complexes thus formed was combined with 20 parts (vol) of HEK293 cells suspended at a density of 2 × 10⁵ per mL, followed by incubation in a humidified 37 °C/5% CO₂ incubator for 20 hr.

Yang X., Smith J.L., Beck M.T., Wilkinson J.M., Michaud A., Vasta J.D., Robers M.B, & Willson T.M. (2023). Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells. Molecules, 28(7), 2950.

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BRET Assay for PLK Engagement

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HEK293 cells (ATCC) were cultured in DMEM (Gibco) + 10% FBS (Seradigm), with incubation in a humidified, 37 °C/5% CO₂ incubator. N-terminal NLuc-PLK1, PLK2 or PLK3 fusions were encoded in pFN31K expression vectors (Promega). Carrier DNA was encoded in pFN5K vectors (Promega). For cellular BRET target engagement experiments, HEK-293 were transfected with NLuc/target fusion constructs using FuGENE HD (Promega) according to the manufacturer’s protocol. Briefly, NLuc/target fusion constructs were diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:9 (mass/mass), after which FuGENE HD was added at a ratio of 1:3 μg DNA: μL FuGENE HD). 1 part (vol) of FuGENE HD complexes thus formed were combined with 20 parts (vol) of HEK-293 cells suspended at a density of 2 × 10⁵ per mL, followed by incubation in a humidified, 37 °C/5% CO₂ incubator for 20 hr.

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iPSA Oxidative Stress Assay Protocol

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HeLa cells were treated with 50 μM Ero1 Inhibitor II-EN460 (Millipore-Merck), prepared in DMSO for 90 min. HeLa cells and iPSAs were treated with 30 μM DMF (dissolved in DMSO) for 48 h. 25 μM BMC (dissolved in methanol; Cayman Chemical) was applied to iPSAs overnight for 18 h. iPSAs were treated with 100 μM hydrogen peroxide (H₂O₂) for 30 min before the assay.
iPSAs were transfected with FugeneHD (Promega) 48 h before performing assays, following the manufacturer’s protocol, using a 1:3 DNA (μg) to FugeneHD (μl) ratio. Neurons were transfected with Lipofectamine 2000, according to the manufacturer’s instruction and experiments were performed 24 h later.
For gene silencing, we used endoribonuclease-prepared siRNA pools (MISSION esiRNA; Sigma-Aldrich), composed of a heterogeneous mixture of siRNAs all targeting the same mRNA sequence. To silence human Nrf2 and SOD1, we used #EHU093471 and #EHU050511, respectively, and #EHURLUC, targeting Renilla Luciferase, was used for the negative control. The cells were transfected with MISSION siRNA transfection reagent, according to the manufacturer’s protocol, for 24 h (Nrf2) or 48 h (SOD1) before the assays.

Granatiero V., Konrad C., Bredvik K., Manfredi G, & Kawamata H. (2019). Nrf2 signaling links ER oxidative protein folding and calcium homeostasis in health and disease. Life Science Alliance, 2(5), e201900563.

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Transient Transfection of 293 Cells

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293FT cells (Invitrogen, Carlsbad, CA, USA) or 293CD4 cells (293 cells constitutively expressing human CD4) [37 (link)] were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were cultured in a 5% CO₂ in a humidified incubator (Sanyo, Japan) and then transferred to 6- or 96-well plates 1 day before transfection, and Fugene HD (Fugene HD [µL]: DNA [µg]: DMEM [µL] = 5:2:200; Promega, Madison, WI, USA) was used for transient transfection. The transfection mix was incubated for 15 min at room temperature prior to addition to the cell culture in a drop-wise manner (10 µL/well). After indicated time after the transfection, the transfected cells were subjected to further analyses, as indicated below.

Liu D., Wang H., Yamamoto M., Song J., Zhang R., Du Q., Kawaguchi Y., Inoue J.I, & Matsuda Z. (2018). Six-helix bundle completion in the distal C-terminal heptad repeat region of gp41 is required for efficient human immunodeficiency virus type 1 infection. Retrovirology, 15, 27.

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Lentiviral Vector Production and Transduction

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Lentiviral constructs were cloned in using the FUGW backbone (Addgene #25870) and packaged in HEK 293T cells by co-transfection with psPAX2 and pCMV-pVSV-G helper plasmids. In brief, 4.4×10⁵ HEK 293T cells per well were seeded in a 6 well plate. After 24-hour incubation, media were replaced with 2mL fresh culture media containing FuGENE HD/DNA complexes. For FuGENE HD/DNA complex, 9 μL of FuGENE HD (Promega) was added to a mixture of 3 plasmids consisting of 0.1 μg of pCMV-VSV-G vector, 0.5 μg of lentiviral packaging psPAX2 vector, and 1 μg of lentiviral expression vector in 100 μL of Opti-MEM reduced serum medium (Thermo Fisher Scientific), followed by 20 minutes incubation at room temperature. Media of transfected cells were replaced with 2 mL of fresh culture media 18 hours post transfection. The supernatant containing newly produced viruses was collected at 48-hours post-transfection, and filtered through a 0.45 μm syringe filter (Pall Corporation, Ann Arbor, MI; Catalog #4614). Filtered lentiviruses were used to infect respective cell lines in the presence of polybrene (Sigma-Aldrich, 8 μg/mL). Successful lentiviral integration was confirmed by using lentiviral plasmid constructs constitutively expressing fluorescent proteins or antibiotic resistance genes to serve as infection markers.

Farzadfard F., Gharaei N., Higashikuni Y., Jung G., Cao J, & Lu T.K. (2019). Single-Nucleotide-Resolution Computing and Memory in Living Cells. Molecular cell, 75(4), 769-780.e4.

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DNA and siRNA Transfection in U2OS Cells

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For DNA U2OS cells were transfected with FugeneHD (Promega) at a DNA:FugeneHD ratio of 1:3.5, according to the manufacturer’s instructions. Before transfection, media was replaced with OptiMEM (Life Technologies), and cells were subsequently incubated with the transfection mix for 4 hours. For transfection in 6-well plates, 1 μg of DNA pr. well was used and scaled accordingly for different well sizes.
For siRNA U2OS cells were reverse transfected with 50 nM On-target Smart Pool siRNA to MSH2 (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen). The mix was then added to newly-seeded cells and medium replaced with complete DMEM after 24 hours. Experiments were performed 48 to 72 hours after transfection.

Nielsen S.V., Stein A., Dinitzen A.B., Papaleo E., Tatham M.H., Poulsen E.G., Kassem M.M., Rasmussen L.J., Lindorff-Larsen K, & Hartmann-Petersen R. (2017). Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations. PLoS Genetics, 13(4), e1006739.

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CRISPR-mediated Ssrp1 tagging in S2R+ cells

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PCR amplification of a sgRNA targeting Ssrp in S2R+ cells for 6Flag integration was done using a combination of oligonucleotides for U6 promoter, Ssrp CRISPR, sgRNA scaffold and sgRNA antisense sequences (Table S2). The pRB17 plasmid (Bottcher et al., 2014 (link)) was used as a template for the U6 promoter sequence during the PCR reaction. A homology repair (HR) donor to integrate a 6Flag-blasticidin cassette at the Ssrp C-terminus was generated by PCR amplification, using oligonucleotides overlapping 60 bp at either side of the stop codon (Table S2). A modified version of the pKF296 plasmid (Kunzelmann et al., 2016 (link)) was used as a template for the HR donor PCR. Both PCR products (sgRNA and HR donor) were purified before transfection using QIAquick PCR purification kit (QIAGEN, 28106). 1.2×10⁵ S2R+ cells bathed 3 days with a combination of dsRNAs against lig4 and mus308 (1 μg/ml each) were seeded in a 96-well plate. A mixture of 50 ng of each purified PCR product and 50 ng pRB14 (plasmid encoding Cas9) (Bottcher et al., 2014 (link)) was transfected with FuGENE HD (Promega, E2311) using a 6:1 ratio (FuGENE HD:DNA). Cells were incubated 4 days with the transfection mix, before being transferred in a 24-well plate in media containing 5 μg/mL blasticidin to select for Ssrp1–6Flag cells.

Jeronimo C., Angel A., Nguyen V.Q., Kim J.M., Poitras C., Lambert E., Collin P., Mellor J., Wu C, & Robert F. (2021). FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner. Molecular cell, 81(17), 3542-3559.e11.

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Knockdown and Ubiquitination Assays in HEK293T Cells

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HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Amarillo, USA) supplemented with 10% (v/v) foetal bovine serum (Assay Matrix, Melbourne, Australia), 2 mM L-glutamine and 100 U mL⁻¹ penicillin/100 µg mL⁻¹ streptomycin (Gibco, Amarillo, TX, USA). Transfections were performed with FuGENE HD (Promega, Madison, WI, USA). For TRIM25 knockdown experiments, HEK293T cells were transfected with 1 µg mL⁻¹ pSuper-shTRIM25 or control pSuper-shScrambled using FuGENE HD (Promega, Madison, WI, USA). For experiments assessing DDX3X ubiquitination, cells were treated with 25 μM MG132 for 4 h prior to harvesting.

Atkinson S.C., Heaton S.M., Audsley M.D., Kleifeld O, & Borg N.A. (2021). TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity. International Journal of Molecular Sciences, 22(16), 9094.

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Transfection of TCam-2 cells

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Transfection of TCam-2 cells with siRNA or a cDNA-coding plasmid was performed as described previously [9 ]. Briefly, 1×10⁵ cells were seeded onto 6-well plates 24h before transfection. For transfection, FuGeneHD (Promega, Mannheim, Germany) was used at a ratio of 5: 1 (e.g. 5 μl FuGeneHD: 1 μg siRNA). ARID1A siRNAs were obtained from Santa Cruz Biotechnology as a pool of three different siRNAs (Santa Cruz, Heidelberg, Germany). Scrambled siRNA was used as negative control and for data normalization (AllStars Negative Control siRNA; Qiagen). siRNA experiments were performed in biological triplicates (n = 3). The GADD45B cDNA-coding plasmid was obtained from Origene (via Biocat, Heidelberg, Germany). The empty vector was used as negative control and for data normalization.

Nettersheim D., Jostes S., Fabry M., Honecker F., Schumacher V., Kirfel J., Kristiansen G, & Schorle H. (2016). A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment. Oncotarget, 7(46), 74931-74946.

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HEK293 Cell Culture and Receptor Expression

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HEK293 (WT) were obtained from the American Type Culture Collection (ATCC), parental HEK293 (WT) and Flp-In T-REx293 were from ThermoFisher. HEK293 (WT), parental and CRISPR/Cas9 genome-edited HEK293 cells were cultured in DMEM (ThermoFisher) containing 10% fetal bovine serum (FBS, PAN biotech), 100 U ml⁻¹ Penicillin, 100 mg ml⁻¹ Streptomycin (ThermoFisher) at 37 °C and 5% CO₂. Receptor expressing HEK293 cell lines were generated by stable transfection of receptor cDNA (GPR17, DP2, β2AR, AT1R, and M3-DREADD cloned into pcDNA3.1(+)) using Fugene HD (Promega) according to manufacturer's instructions and subsequently cultivated using growth medium containing 500 µg ml⁻¹ G418 (Invivogen). FFA2 receptor was expressed in Flp-In T-REx293 cells upon induction of expression after treatment with 1 µg ml⁻¹ doxycycline for at least 16 h. Transiently transfected cells were analyzed 24–48 h after transfection using Fugene HD (Promega). All cell lines were checked for and free of mycoplasma contamination. All chemicals were purchased from Sigma-Aldrich unless otherwise indicated. For G protein inhibition, cells were incubated with pertussis toxin (PTX) for at least 18 h at 150 ng ml⁻¹ and FR for at least 1 h at 1 µM.

Grundmann M., Merten N., Malfacini D., Inoue A., Preis P., Simon K., Rüttiger N., Ziegler N., Benkel T., Schmitt N.K., Ishida S., Müller I., Reher R., Kawakami K., Inoue A., Rick U., Kühl T., Imhof D., Aoki J., König G.M., Hoffmann C., Gomeza J., Wess J, & Kostenis E. (2018). Lack of beta-arrestin signaling in the absence of active G proteins. Nature Communications, 9, 341.

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