17-10404 LentiBrite™ RFP-p62 Lentiviral Biosensor

17-10404
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 Fluorescent
      Description
      Catalogue Number17-10404
      Trade Name
      • LentiBrite
      • Chemicon
      DescriptionLentiBrite™ RFP-p62 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™ RFP-p62 lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of autophagy in difficult-to-transfect cell types.
      Alternate Names
      • Sequestosome-1
      • Sequestosome
      • Ubiquitin-binding protein p62
      • EBI3-associated protein
      • p62
      Background InformationAutophagy, a degradative pathway induced in cells under stress, plays both protective and deleterious roles in many diseases, including cancer, neurodegeneration, and infections. The adaptor protein p62 targets protein aggregates to the autophagosome for degradation via its polyubiquitin-binding domain, and also functions as a scaffold for signaling proteins such as atypical PKCs and mTORC1. In many cell types, p62 ordinarily exists as puncta or speckles, which increase in number and size upon blockade of autophagic flux with lysosome inhibitors. Upon initiation of autophagy, the p62-cargo protein complex binds to LC3 on the autophagosome surface, which results in degradation of this complex. DNA constructs encoding fluorescent proteins fused to p62 are often introduced into cells for monitoring aggregate formation by fluorescence microscopy. EMD Millipore’s LentiBriteTM RFP-p62 lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of autophagy in difficult-to-transfect cell types.
      References
      Product Information
      Components
      • TagRFP-p62 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 lot specific “Viral Titer” in the product specifications of 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, HeLa, U2OS, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC)).
        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
      Application NotesFluorescence Microscopy
      Imaging:
      (See Figure 1 in datasheet)
      Primary cell type, Human mesenchymal stem cells (HuMSC), were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 24 hours further incubation, cells were either left in complete media or incubated for 4 hours in EBSS containing a lysosome inhibitor, to induce autophagy and inhibit lysosomal degradation of autophagosomes. Cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy. The RFP-p62 displays a diffuse cytosolic distribution with scattered small puncta in fed cells, and an entirely punctate distribution in starved cells.


      Immunocytochemistry Comparison:
      (See Figure 2 in datasheet)
      Similar to Figure 1 (see datasheet), U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 24 hours. Cells were then fixed and stained with a polyclonal antibody against p62 (Cat. No. MABC32) followed by a FITC-labeled anti-rabbit IgG. Distribution of the RFP-p62 (red) is similar to that detected by immunocytochemical staining (green).

      Hard-to-transfect Cell Types:
      (See Figure 3 in datasheet)
      Primary cell type HUVEC were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. Subsequent treatments for cells left in complete media or cells incubated in EBSS with lysosome inhibitor, were performed as in Figures 1A and 1B (see datasheet)

      Time-lapse Imaging:
      (See Figure 5 in datasheet and video online)
      U2OS cells were plated in coverglass chamber slides and transduced with RFP-p62 lentiviral particles as in Figure 1 (see datasheet). After replacing growth media with EBSS containing a lysosomal inhibitor, images were collected every 20 seconds for a total of 32 min. The RFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

      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
      • p62
      • SQSTM1
      • ORCA
      • OSIL
      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 MilliporeSigma 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

      Documentation

      LentiBrite™ RFP-p62 Lentiviral Biosensor SDS

      Title

      Safety Data Sheet (SDS) 

      LentiBrite™ RFP-p62 Lentiviral Biosensor Certificates of Analysis

      TitleLot Number
      LentiBrite RFP-p62 Lentiviral Biosensor - Q2068465Q2068465
      LentiBrite™ RFP-p62 -25262132526213
      LentiBrite™ RFP-p62 -25563712556371
      LentiBrite™ RFP-p62 -27260842726084
      LentiBrite™ RFP-p62 -27300142730014
      LentiBrite™ RFP-p62 -27497172749717
      LentiBrite™ RFP-p62 -27578622757862
      LentiBrite™ RFP-p62 Lentiviral Biosensor Packaged Lentiviral Particles2966781
      LentiBrite™ RFP-p62 Lentiviral Biosensor Packaged Lentiviral Particles2990706
      LentiBrite™ RFP-p62 Lentiviral Biosensor Packaged Lentiviral Particles2918433

      Brochure

      Title
      Hallmarks of Aging (EMD)

      Posters

      Title
      Poster: Autophagy Signaling