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X tremegene 9 transfection reagent

Manufactured by Merck Group
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The X-tremeGENE 9 transfection reagent is a lipid-based formulation designed for efficient and reliable transfection of various cell types, including hard-to-transfect cells. The reagent enables the delivery of DNA, RNA, and other molecules into the target cells.

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Lab products found in correlation

Dual glo reagent, by Promega (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions) Opti mem, by Thermo Fisher Scientific (4 mentions) Polybrene, by Santa Cruz Biotechnology (3 mentions) Pspax2, by Addgene (3 mentions) Dh10bac e coli, by Thermo Fisher Scientific (3 mentions) Ni nta superflow nickle charged resin, by Qiagen (3 mentions) Pfastbac1 vector, by Thermo Fisher Scientific (3 mentions) Dual luciferase reporter 1000 assay system, by Promega (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Plenti pgk blast v5 luc w528 1, by Addgene (2 mentions) Lenticrispr v2 vector, by Addgene (2 mentions) Pcmv vsv g, by Addgene (2 mentions) Amicon ultra 15 centrifuge filter, by Merck Group (2 mentions) Pmd2 g, by Addgene (2 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Ca stat3, by Addgene (1 mentions) Protein g sepharose, by GE Healthcare (1 mentions)

50 protocols using x tremegene 9 transfection reagent

1

Lentiviral Transduction in HEK-293T Cells

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HEK-293T cells were co-transfected with the pcw107-V5 plasmid expressing CA-STAT3 (Addgene, (Watertown, MA, USA), #64611), VSV-G envelope plasmid, and Δvpr lentiviral plasmid using X-TremeGene 9 Transfection Reagent (MERCK, # 6365779001). The supernatant containing the virus was collected 48 h after transfection and spun for 5 min at 400 g to eliminate cells.
Solaimuthu B., Lichtenstein M., Hayashi A., Khatib A., Plaschkes I., Nevo Y., Tanna M., Pines O, & Shaul Y.D. (2022). Depletion of Fumarate Hydratase, an Essential TCA Cycle Enzyme, Drives Proliferation in a Two-Step Model. Cancers, 14(22), 5508.
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2

Overexpression of FLAG-tagged PREPL

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Generation of FLAG-tagged PREPL constructs for overexpression in mammalian cells has been described before (12) . HEK293T cells were grown in DMEM supplemented with 10% FCS at 37°C, 5% CO2 and transfected using the Xtreme Gene 9 transfection reagent (Merck) according to the manufacturer's protocol. 24 hours post-transfection, cells were lysed by passaging through a 26-gauge needle. Cell debris was removed by centrifugation at 16000 g for 10 minutes. FLAG-tagged proteins were immunopurified using preformed complexes of anti-FLAG M2 antibody (Sigma-Aldrich) and protein G Sepharose (GE healthcare).
Monnens Y., Theodoropoulou A., Rosier K., Bhalla K., Mahy A., Vanhoutte R., Meulemans S., Cavani E., Antanasijevic A., Lemmens I., Lee J.A., Spellicy C.J., Schroer R.J., Maselli R.A., Laverty C.G., Agostinis P., Pagliarini D.J., Verhelst S., Marcaida M.J., Rochtus A., Dal Peraro M, & Creemers J.W. (2024). Missense variants in CMS22 patients reveal that PREPL has both enzymatic and non-enzymatic functions. JCI insight.
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3

HeLa and HEK293 cell culture protocols

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Human HeLa cells (kindly provided by Melchior F., Universität Heidelberg, Germany), HEK293 Flp-In T-REx cells (Thermo Fischer Scientific, Waltham, MA, USA), and HeLa Flp-In T-REx cells (kindly provided by Mayer T., Universität Konstanz, Germany) were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin–streptomycin (Biosera, Nuaille, France). For hypoxic treatment, cells were exposed to 1% O2, and 5% CO2 in an INVIVO2 200 hypoxia workstation (Ruskinn Life Sciences, Pencoed, UK) for the indicated times. When required, cells were treated with 2 μΜ rapamycin for 6 h (Sigma-Aldrich, St Louis, MO, USA) or DMSO at the appropriate concentration as a solvent control.
HeLa Flp-In T-REx cells were transfected with pcDNA5-2xFlag-His6-siinsEXOSC10 (WT or K583R) or pcDNA5-2xFlag-His6 (empty vector) plasmids using XtremeGene9® transfection reagent (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s guidelines. Transfected cells in which the transgene was integrated into the Flp-In locus were selected with 1 µg/mL of blasticidin and 200 µg/mL of hygromycin.
Filippopoulou C., Thomé C.C., Perdikari S., Ntini E., Simos G., Bohnsack K.E, & Chachami G. (2024). Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile. Cellular and Molecular Life Sciences: CMLS, 81(1), 58.
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4

Quantifying IFNα2 and IFNω Autoantibody Blocking

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The blocking activity of anti-IFNα2 and anti-IFNω autoantibodies was determined with a reporter luciferase activity as described previously64 . HEK293T cells were transfected with a plasmid containing the firefly luciferase gene under the control of the human ISRE promoter in the pGL4.45 backbone and a plasmid constitutively expressing Renilla luciferase for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGENE9 transfection reagent (Sigma-Aldrich, 6365779001) for 24 h. Cells in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 2% fetal calf serum and 10% healthy control or patient serum/plasma (after inactivation at 56 °C for 20 min) were stimulated with IFNα2 (Miltenyi Biotec, 130-108-984) and IFNω (Merck, SRP3061) at 10 ng ml−1 or 100 pg ml−1 for 16 h at 37 °C. Each sample was tested once for each cytokine and dose. Finally, cells were lysed for 20 min at room temperature, and luciferase levels were measured with the Dual-Luciferase Reporter 1000 Assay System (Promega, E1980) according to the manufacturer’s protocol. Luminescence intensity was measured with a VICTOR-X Multilabel Plate Reader (PerkinElmer Life Sciences). Firefly luciferase activity values were normalized against Renilla luciferase activity values.
Malle L., Patel R.S., Martin-Fernandez M., Stewart O.J., Philippot Q., Buta S., Richardson A., Barcessat V., Taft J., Bastard P., Samuels J., Mircher C., Rebillat A.S., Maillebouis L., Vilaire-Meunier M., Tuballes K., Rosenberg B.R., Trachtman R., Casanova J.L., Notarangelo L.D., Gnjatic S., Bush D, & Bogunovic D. (2023). Autoimmunity in Down’s syndrome via cytokines, CD4 T cells and CD11c+ B cells. Nature, 615(7951), 305-314.
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5

Optimized Transfection of HEK293T Cells

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These experiments all utilized HEK293T cells passaged in DMEM media with 10% FBS. Transfection growth was done on 8-well glass slides (CellVis) previously coated with 0.1 mg/mL poly-D-lysine (Sigma). After reaching approximately 90% confluency, cells were resuspended in DMEM via trypsin-EDTA digestion and counted using a haemocytometer. The cells were then diluted to an approximate concentration of 250,000 to 300,000 cells/mL – a relatively low value, in order to ensure a clean monolayer for visualization. 250 μL of cells were added to each well. After an initial adherence and growth period of 20 hours, the cells were transfected with X-tremeGENE 9 transfection reagent (Sigma): 50 μL master mixes were made by combining 1 μL of a reporter construct at 50 ng/uL and 5 μL of an editor construct at 100 ng/μL with 44 μL of OPTI-mem reduced serum media (Thermo Fisher), adding 1.5 μL of reagent, and allowing it to sit at room temperature for 30 minutes. 15 μL of each mix was then added dropwise to a particular well, and expression was allowed to occur for 48 hours at 37C, 5% CO2.
Wolfe A.D., Arnold D.B, & Chen X. (2019). Comparison of RNA editing activity of APOBEC1-A1CF and APOBEC1-RBM47 complexes reconstituted in HEK293T cells. Journal of molecular biology, 431(7), 1506-1517.
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6

Virus Generation from DNA Clones

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To generate virus from DNA-based infectious clones, 3 μg of plasmid were transfected into 2 individual 6 wells of 400,000 BHK-21J cells using X-treme Gene9 Transfection Reagent (Sigma). Three days post transfection, the supernatant from 2 individual wells was combined and 3 ml was transferred to a T25 flask containing 1 x 106 VeroE6-C1008-TMPRSS2 cells and 2 ml serum-free MEM. After 4 days, 250 μl supernatant was added to 750 μl TriReagent for RNA extraction and RT-qPCR. T25 flasks were fixed with 4% paraformaldehyde, imaged on Nikon Eclipse Ti and processed with ImageJ.
Park G.J., Osinski A., Hernandez G., Eitson J.L., Majumdar A., Tonelli M., Henzler-Wildman K., Pawłowski K., Chen Z., Li Y., Schoggins J.W, & Tagliabracci V.S. (2022). The mechanism of RNA capping by SARS-CoV-2. Research Square.
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7

Quantifying Interferon Neutralization by Auto-Abs

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The blocking activity of auto-Abs against IFN-α2 and IFN-ω was also determined by assessing reporter luciferase activity. Briefly, HEK293T cells were transfected with the firefly luciferase plasmids under the control human ISRE promoters in the pGL4.45 backbone, and a constitutively expressing Renilla luciferase plasmid for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGene 9 transfection reagent (Sigma-Aldrich) for 36 h. DMEM (Thermo Fisher Scientific) was supplemented with 10% healthy control or patient serum/plasma and was either left unstimulated or was stimulated with IFN-α2 and IFN-ω (10 ng/ml) for 16 h at 37°C. Finally, luciferase levels were measured with the Dual-Glo reagent according to the manufacturer’s protocol (Promega). Firefly luciferase values were normalized against Renilla luciferase values.
Bastard P., Orlova E., Sozaeva L., Lévy R., James A., Schmitt M.M., Ochoa S., Kareva M., Rodina Y., Gervais A., Le Voyer T., Rosain J., Philippot Q., Neehus A.L., Shaw E., Migaud M., Bizien L., Ekwall O., Berg S., Beccuti G., Ghizzoni L., Thiriez G., Pavot A., Goujard C., Frémond M.L., Carter E., Rothenbuhler A., Linglart A., Mignot B., Comte A., Cheikh N., Hermine O., Breivik L., Husebye E.S., Humbert S., Rohrlich P., Coaquette A., Vuoto F., Faure K., Mahlaoui N., Kotnik P., Battelino T., Trebušak Podkrajšek K., Kisand K., Ferré E.M., DiMaggio T., Rosen L.B., Burbelo P.D., McIntyre M., Kann N.Y., Shcherbina A., Pavlova M., Kolodkina A., Holland S.M., Zhang S.Y., Crow Y.J., Notarangelo L.D., Su H.C., Abel L., Anderson M.S., Jouanguy E., Neven B., Puel A., Casanova J.L, & Lionakis M.S. (2021). Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1. The Journal of Experimental Medicine, 218(7), e20210554.
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8

Overexpression and knockdown of key signaling proteins

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The pcDNA3.1-NOX4 and pUSE-Akt1CA plasmids have been previously described (11 , 23 (link)). Cells were transfected using X-treme GENE 9 Transfection Reagent (Sigma) according to the manufacturer's protocol. Cells were transfected with 100 nM scramble, human Akt1, human NOX4, or mouse Bad siRNA duplex (IDT) utilizing Dharmafect 2 (Thermo Scientific) according to manufacturer's protocol. Eight hours after transfection, media was replaced, and cells were allowed to recover for 24–72 h.
Larson-Casey J.L., Gu L., Kang J., Dhyani A, & Carter A.B. (2021). NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression. The Journal of Biological Chemistry, 297(1), 100810.
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9

Stable transfection of HEK293 cells

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Parental HEK293 cells were cultivated in DMEM (high glucose) supplemented with 10 % (v/v) fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate 100 U/mL penicillin and 100 μg/mL streptomycin. One day after transfection of 106 parental HEK293 cells in a 6-well plate with 1 μg plasmid encoding C-terminally FLAG-HA-tagged carriers using X-tremeGENE 9 transfection reagent (Merck Sigma XTG9-RO), one fifth of the cells were distributed onto a 10 cm dish and grown for ten days in complete DMEM supplemented with 550 μg/mL G418 whilst replacing the medium every other day. A total of 350 surviving cells were seeded on a 10 cm dish for a first round of clonal selection. Subsequently, cell clones were transferred into a 24 well plate and further expanded. Cells stably expressing the transgene were identified by FLAG-immunoblot analysis and subjected to another round of clonal selection.
Luongo T.S., Eller J.M., Lu M.J., Niere M., Raith F., Perry C., Bornstein M.R., Oliphint P., Wang L., McReynolds M.R., Migaud M.E., Rabinowitz J.D., Johnson F.B., Johnsson K., Ziegler M., Cambronne X.A, & Baur J.A. (2020). SLC25A51 is a mammalian mitochondrial NAD+ transporter. Nature, 588(7836), 174-179.
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10

Assessing Anti-IFN Autoantibody Blocking Activity

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The blocking activity of anti-IFN-α and anti-IFN-ω autoantibodies was determined by assessing a reporter luciferase activity. Briefly, HEK293T cells were transfected with the firefly luciferase plasmids under the control human ISRE promoters in the pGL4.45 backbone, and a constitutively expressing Renilla luciferase plasmid for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGene 9 transfection reagent (Sigma Aldrich) for 36 hours. Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific) was supplemented with 10% healthy control or patient serum or plasma and was either stimulated with IFN-α or IFN-ω (10 ng/mL), or left unstimulated for 16 hours at 37°C. Each sample was tested once. Finally, luciferase expression were measured with the Dual-Glo reagent, according to the manufacturer’s protocol (Promega). Firefly luciferase values were normalized against Renilla luciferase values, and fold induction was calculated relative to controls transfected with empty plasmids.
van der Wijst M.G., Vazquez S.E., Hartoularos G.C., Bastard P., Grant T., Bueno R., Lee D.S., Greenland J.R., Sun Y., Perez R., Ogorodnikov A., Ward A., Mann S.A., Lynch K.L., Yun C., Havlir D.V., Chamie G., Marquez C., Greenhouse B., Lionakis M.S., Norris P.J., Dumont L.J., Kelly K., Zhang P., Zhang Q., Gervais A., Le Voyer T., Whatley A., Si Y., Byrne A., Combes A.J., Rao A.A., Song Y.S., Fragiadakis G.K., Kangelaris K., Calfee C.S., Erle D.J., Hendrickson C., Krummel M.F., Woodruff P.G., Langelier C.R., Casanova J.L., Derisi J.L., Anderson M.S, & Ye C.J. (2021). Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Science Translational Medicine, 13(612), eabh2624.
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