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17-10208 | LentiBrite™ EB3-GFP Lentiviral Biosensor

17-10208
1 vial (minimum of 3 x 10E8 IFU/mL)  
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      Overview

      Replacement Information

      Key Specifications Table

      Key ApplicationsDetection Methods
      TFX, IF, ICC, Track single cell and cell population migration (migration assay, cell culture) Fluorescent
      Description
      Catalogue Number17-10208
      Trade Name
      • LentiBrite
      • Chemicon
      DescriptionLentiBrite™ EB3-GFP Lentiviral Biosensor
      OverviewRead our application note in Nature Methods!
      http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf
      (Click Here!)

      Learn more about the advantages of our LentiBrite Lentiviral Biosensors! Click Here

      Biosensors can be used to detect the presence/absence of a particular protein as well as the subcellular location of that protein within the live state of a cell. Fluorescent tags are often desired as a means to visualize the protein of interest within a cell by either fluorescent microscopy or time-lapse video capture. Visualizing live cells without disruption allows researchers to observe cellular conditions in real time.

      Lentiviral vector systems are a popular research tool used to introduce gene products into cells. Lentiviral transfection has advantages over non-viral methods such as chemical-based transfection including higher-efficiency transfection of dividing and non-dividing cells, long-term stable expression of the transgene, and low immunogenicity.

      EMD Millipore is introducing LentiBrite™ Lentiviral Biosensors, a new suite of pre-packaged lentiviral particles encoding important and foundational proteins of autophagy, apoptosis, and cell structure for visualization under different cell/disease states in live cell and in vitro analysis.
      • Pre-packaged, fluorescently-tagged with GFP & RFP
      • Higher efficiency transfection as compared to traditional chemical-based and other non-viral-based transfection methods
      • Ability to transfect dividing, non-dividing, and difficult-to-transfect cell types, such as primary cells or stem cells
      • Non-disruptive towards cellular function

      EMD Millipore’s LentiBrite™ EB3-GFP lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of microtubule plus-end dynamics in difficult-to-transfect cell types.
      Background InformationMicrotubules are dynamic cytoskeletal filaments comprising a core of tubulin subunits flanked by structurally and functionally distinct termini, termed the plus-end and minus-end. The dynamic instability of microtubules, consisting of cycles of growth and decay, occurs at the plus-ends and is mediated by a complex of microtubule plus-end tracking proteins (+TIPs). Key components of the +TIP complex are the end-binding proteins (EBs). Imaging of plus-end growth in live cells expressing EBs tagged with fluorescent proteins has contributed greatly to our understanding of microtubule dynamics.
      EMD Millipore’s LentiBrite™ EB3-GFP lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of microtubule plus-end dynamics in difficult-to-transfect cell types.
      References
      Product Information
      Components
      • EB3-TagGFP2 Lentivirus:
        One vial containing 25 µL of lentiviral particles at a minimum of 3 x 10E8 infectious units (IFU) per mL. For lot-specific titer information, please see “Viral Titer” in the datasheet.

      • Promoter
        EF-1 (Elongation Factor-1)
      • Multiplicty of Infection (MOI)
        MOI = Ratio of # of infectious lentiviral particles (IFU) to # of cells being infected.
        Typical MOI values for high transduction efficiency and signal intensity are in the range of 20-40. For this target, some cell types may require lower MOIs (e.g., HT-1080, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), U2OS, HeLa).
        NOTE: MOI should be titrated and optimized by the end user for each cell type and lentiviral target to achieve desired transduction efficiency and signal intensity.
      Detection methodFluorescent
      Applications
      Key Applications
      • Transfection
      • Immunofluorescence
      • Immunocytochemistry
      • Track single cell and cell population migration (migration assay, cell culture)
      Application NotesFluorescence Microscopy Imaging:
      REF52 cells were plated in a chambered coverglass and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were imaged live by oil immersion wide-field fluorescence microscopy. The EB3-GFP displays a punctate distribution characteristic of microtubule ends.

      Immunocytochemistry Comparison and Inhibitor Analysis:
      HeLa cells were plated in a coverglass chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. EB3-GFP displays a nearly complete overlap with EB3 detected by immunocytochemical staining with rabbit polyclonal anti-EB3 (EMD Millipore cat. no. AB6033).

      Additional Cell Types:
      MCF7 cells were transduced with EB3-RFP lentivirus as in Figure 1 at an MOI 40 for 24 hours, then stained for tubulin. EB3-GFP displays a punctate and short linear distribtion. Immunocytochemical staining of the same field of view with a monoclonal antibody against α-tubulin (EMD Millipore Cat. No. 05-829) reveals to the typical filamentous pattern of microtubules. Overlay of EB3-GFP (green), anti-tubulin (red) and DAPI (blue) demonstrates that EB3-GFP is localized to the microtubule ends.

      Hard-to-transfect Cell Types:
      Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in coverglass chamber slides and transduced with EB3-GFP lentiviral particles at an MOI of 20 and 40, respectively, for 24 hours.

      Time-lapse Imaging:
      REF52 cells were plated in coverglass chamber slides and transduced with EB3-GFP lentiviral particles. Images were collected every 3 seconds for a total of 2 min. Shown here are 3 sequential frames; to see the entire sequence, please visit our Cell Structure-Biosensors page.

      For optimal fluorescent visualization, it is recommended to analyze the target expression level within 24-48 hrs after transfection/infection for optimal live cell analysis, as fluorescent intensity may dim over time, especially in difficult-to-transfect cell lines. Infected cells may be frozen down after successful transfection/infection and thawed in culture to retain positive fluorescent expression beyond 24-48 hrs. Length and intensity of fluorescent expression varies between cell lines. Higher MOIs may be required for difficult-to-transfect cell lines.
      Biological Information
      Gene Symbol
      • MAPRE3
      Purification MethodPEG precipitation
      UniProt Number
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceEvaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of transduction efficiency.
      Usage Statement
      • This product contains genetically modified organisms (GMO). Within the EU GMOs are regulated by Directives 2001/18/EC and 2009/41/EC of the European Parliament and of the Council and their national implementation in the member States respectively. This legislation obliges EMD Millipore to request certain information about you and the establishment where the GMOs are being handled. Click here for Enduser Declaration (EUD) Form.
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStorage and Handling
      Lentivirus is stable for at least 4 months from date of receipt when stored at -80°C. After first thaw, place immediately on ice and freeze in working aliquots at -80°C. Frozen aliquots may be stored for at least 2 months. Further freeze/thaws may result in decreased virus titer and transduction efficiency.

      IMPORTANT SAFETY NOTE
      Replication-defective lentiviral vectors, such as the 3rd Generation vector provided in this product, are not known to cause any diseases in humans or animals. However, lentiviruses can integrate into the host cell genome and thus pose some risk of insertional mutagenesis. Material is a Risk Group 2 and should be handled under BSL2 controls. A detailed discussion of biosafety of lentiviral vectors is provided in Pauwels, K. et al. (2009). State-of-the-art lentiviral vectors for research use: Risk assessment and biosafety recommendations. Curr. Gene Ther. 9: 459-474.
      Packaging Information
      Material Size1 vial (minimum of 3 x 10E8 IFU/mL)
      Transport Information
      Supplemental Information
      Specifications