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  • TransPass™ V

    Discontinued Date

    01/01/2013
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    Discontinued Products

    Description

    TransPass™ V is an Adenovirus-derived enhancer component that is used along with a transfection reagent. The combination of a TransPass transfection reagent & TransPass-V significantly enhances transfection efficiency in many cell lines and primary cells including endothelial or epithelial (1,2). For example, the combination of TransPass D2 & TransPass V yields optimal plasmid transfection efficiency in suspension cells (Figure 1). This combination has low toxicity and thus, transfected cells show very little, if any, cell death (Figure 2).

    *TransPass-V contains a replication-deficient Adenovirus preparation. Because of the nature of this component, it should not be used with cell lines that contain Adenovirus sequences such as HEK-293, to avoid complementation of the virus. Additionally, it is recommended that common laboratory biosafety used in standard Adenovirus work is practiced. For more information see http://oba.od.nih.gov/oba/index.html.

    TransPass V is a proprietary formulation manufactured by Targeting Systems. Please direct all inquirires regarding reagent composition to Targeting Systems.

    Cell Lines Successfully Transfected:
    • MEF (TransPass D2 & TransPass V)
    • IMR-90 (TransPass D2 & TransPass V)
    • HepG2 (TransPass D2 & TransPass V)
    • Huh-7 (TransPass D1, TransPass D2 & TransPass V)
    • MDCK (TransPass D1, TransPass D2 & TransPass V)
    • A549 (TransPass R1 & TransPass V)
    • T47D (TransPass R1 & TransPass V)
    • Jurkat (TransPass D2, TransPass R1 & TransPass V) (Figure 1)
    Guidelines for Endothelial Cell Culture:
    • Use early passages of cells (up to the 6th passage) for transfections.
    • Use non-collagen and non-gelatin coated tissue culture plates only (transfection of cells plated on collagen-coated surfaces may result in lower transfection efficiency).
    • Recommended media: MCDB131 from VEC technologies, Media 199 plus supplements and 20% fetal calf serum or EBM from Cambrex plus supplements and 10% fetal calf serum. Cells grown in these media appear healthier and give higher transfection efficiency.
    • Clonetics HUVEC cells have given very good transfection results.
    Transfection Guidelines:
    • For consistent results, it is important to maintain healthy proliferating cells that are regularly passaged.
    • If cells have been grown in medium containing heparin, they must be washed after trypsinizing and resuspended in growth medium without heparin and antibiotics/antimycotics before plating for transfection.
    • It is important that NO heparin and NO antibiotics/antimycotics in the growth medium during transfection.
    • Use sterile plasmid DNA purified by CsCl gradient centrifugation or column chromatography.
    • The amount of plasmid DNA per transfection can be varied, and the ratio of HUVEC Reagent Component to TransPass-V should be kept between (1:1) and (1:2).
    Figure 1:
    Transfection efficiency of TransPass reagents in Jurkat. Although Jurkat is a suspension cell line, the proliferating culture of Jurkat, was trypsinized to declump the cells. Trypsin was added to cell pellet followed by 5 minutes of incubation at 37°C, 5% CO2. The cells were washed once and resuspended in complete growth medium containing no antibiotics/antimycotics before plating at 70% cell density for transfection. Transfection was carried out for 24 hours. Supernatants were collected and assayed for Gaussia Luciferase (GLuc) activity using the Gaussia Luciferase Assay Kit (NEB #E3300).
    Figure 2:
    Transfection of Jurkat cells with Fluorescein-siRNA Transfection Control (NEB #N2100 ) using a combination of TransPass D2 (NEB #M2554 ) & TransPass V. Cells were fixed and permeabilized (A) FITC, (B) DAPI.


    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. Transfection complex mixture must be made in serum-free media.
    2. For a transfection complex mixture containing plasmid(s) and/or siRNA, the plasmids and/or siRNA should be mixed well together before adding them to the serum-free media, followed by the TransPass reagents.
    3. The mixture of plasmid(s) and/or siRNA & a TransPass transfection reagent in serum-free media should be incubated at room temperature for 25-30 minutes before adding TransPass V.
    4. Recommended ratios of TransPass transfection reagent & TransPass V are 1:1, 1:2 & 1:3.
    5. For transfection, it is important that cells are resuspended & plated out in complete growth media containing no antibiotics/antimycotics.
    6. It is important that transfection is carried out in non-coated vessels (plates, flasks, etc.).

    References

    1. Stecenko, A. et al. (2000). Exp. Lung Res.. 26, 179-201.
    2. Raja-Walia, R. et al. (1995). Gene Ther.. 2, 521-530.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.
    1. Cellular Imaging and Analysis FAQs
    1. Combination of TransPass D1 or TransPass D2 & TransPass V (M2561)

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