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Jetmessenger

Manufactured by Polyplus Transfection
Sourced in France

JetMessenger is a lab equipment product designed for the transfection of nucleic acids into mammalian cells. It facilitates the delivery of DNA, RNA, or other molecules into the target cells.

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5 protocols using jetmessenger

1

Western Blot Analysis of Recombinant Protein

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mRNA was transfected into HEK293 cells using a transfection reagent (jetMESSENGER™; PolyPlus, Brant, France). After 24 h, total cell protein and whole-cell supernatants were collected. Each lysate sample underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels using TRIS-HCl. Then, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were enclosed in TBST (Tris-buffered saline containing Tween 20) solution with 5% skimmed milk. PVDF membranes were treated with primary (rabbit anti-VP7) antibody, followed by addition of a secondary antibody (horseradish peroxidase-coupled goat anti-rabbit IgG; Abcam, Cambridge, UK), followed by incubation for 1 h at room temperature. After washing, treated PVDF membranes were exposed to a highly sensitive luminescence solution (PK10003; Proteintech, Chicago, IL, USA) and imaged using an electrochemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA).
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2

Optimized iodixanol-based RNA transfection

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OptiPrep, a 60% (w/v) solution of iodixanol, was obtained from Axis-Shield (Dundee, Scotland). The FAM-labeled A-NA RNA oligomer 5′-ACGCGCGCGCGUU UUCGCGCGCGCGU-3′ was synthesized by Tianjin Biolino Acid Technology (Tianjin, China). JetMessenger, an RNA transfection reagent, was purchased from Polyplus (Illkirch, FRANCE). NOHA was obtained from Sigma (Beijing, China). The compound 1400W was purchased from Apex Bio (Houston, Unite States). Polyvinyl alcohol (P139535) was obtained from Aladdin (Shanghai, China). The antibodies used for Western blotting and ELISAs are described in Supplemental Table 1.
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3

RNA Transfection of HAP1 Cells

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HAP1 cells were transfected with 30 pmol RNA fragments with JetMESSENGER (150-001, Polyplus, Illkirch, France) according to the manufacturer’s protocol. After 4 h of incubation, the media was changed to fresh new media, and the cells were subsequently used for ICC or cell viability assay.
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4

Optimizing mRNA Delivery with Cationic Nanoemulsions

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DC2.4 (1×105 cells/well) were seeded in a 24-well plate 24 h before transfection. Then, CNEs (10%, 20%, or 30% DOTAP) were incubated with 0.75 μg of enhanced GFP-encoding mRNA (5moU) (Cat#: L-7201, TriLink Biotechnologies, San Diego, CA) to reach a N/P ratio ranging from 1.5 to 54 in a total volume of 50 μL DEPC-treated water respectively for 30 min at 4 °C, while 1 μg of JetMessenger (Polyplus-transfection® SA, New York, USA) was used as positive control. Meanwhile, the transfection efficiency of mRNA-loaded CNEs (30% DOTAP, N/P = 4.5) prepared by electrostatic adsorption or Microfluidic Chip (Micronit, X3550 CH.2, Netherlands) on both BMDCs and DC2.4 was also investigated. Later, 50 μL carrier-mRNA complexes were added to each well containing 450 μL serum-free DMEM medium. Six hours later, the RPMI 1640 medium in the wells was replaced with fresh medium containing 10% FBS. Another 18 h later, cells were washed twice with PBS and imaged under a microscope, with fluorescence pictures captured and further analyzed.
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5

Formulation of Lipid Nanoparticles for Delivery

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Gelatin GELITA® MedellaPro® < 100, porcine gelatin, 228 g Bloom, pharmaceutical-grade was purchased from GELITA® Deutschland GmbH, Eberbach, Germany. Protamine sulfate was purchased from Sigma-Aldrich, Darmstadt, Germany. JetMessenger (JetM) and JetPrime (JetP) were purchased from Polyplus-transfection®, Illkirch, France. Branched polyethyleneimine (PEI), Mw~25,000, was purchased from Sigma-Aldrich Darmstadt, Germany. Lipofectin was purchased from Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany. Purified water was obtained from a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany), and is referred to as MQ water. DLin-MC3-DMA was purchased from MedChemExpress (Middlesex County, NJ, USA), Cholesterol was purchased from Sigma-Aldrich Darmstadt, Germany, DSPE-PEG 2000 and DPPC were a kind gift from Lipoid GmbH, Ludwigshafen, Germany.
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