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17-10188 LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Key Applications: Transfection, Immunofluorescence, Immunocytochemistry
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17-10188
1 vial (minimum of 3 x 10E8 IFU/mL)  
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      Overview

      Replacement Information

      Key Specifications Table

      Key ApplicationsDetection Methods
      TFX, IF, ICCFluorescent
      Description
      Catalogue Number17-10188
      Trade Name
      • LentiBrite
      • Chemicon
      DescriptionLentiBrite™ RFP-LC3 Control Mutant 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-LC3-G120A lentiviral particles serve as a negative control alongside RFP-LC3 wild-type (catalog # 17-10143) for live cell analysis of autophagy.
      Alternate Names
      • Microtubule-associated proteins 1A/1B light chain 3
      • Autophagy-related protein LC3
      • Autophagy-related ubiquitin-like modifier LC3
      • MAP1A/MAP1B light chain 3
      • Microtubule-associated protein 1 light chain 3
      Background InformationAutophagy, a stress-induced degradative pathway, plays a role in many diseases, including cancer, neurodegeneration, and infections. Members of the LC3 family play a key role in the maturation of the autophagosome, the central organelle of autophagy. LC3 precursors are proteolytically processed to form cytosolic LC3-I. Upon initiation of autophagy, the C-terminal glycine is modified by addition of phosphatidylethanolamine (PE) to form LC3-II, which translocates to autophagosomes in a punctate distribution. DNA constructs encoding fluorescent proteins fused to LC3 are widely employed for monitoring autophagosome formation by fluorescence microscopy.
      A mutant form of LC3 with the C-terminal glycine changed to alanine (LC3-G120A) is unable to accept the PE modification and fails to translocate to the autophagosome upon induction of autophagy.
      EMD Millipore’s LentiBrite™ RFP-LC3-G120A lentiviral particles serve as a negative control alongside RFP-LC3 wild-type (catalog # 17-10143) for live cell analysis of autophagy.
      References
      Product Information
      Components
      • TagRFP-LC3-G120A 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), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), human mesenchymal stem cells (HuMSC), U2OS).
        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
      Quality LevelMQ100
      Applications
      Key Applications
      • Transfection
      • Immunofluorescence
      • Immunocytochemistry
      Application NotesFluorescence Microscopy Imaging:
      (See Figure 1 in datasheet)
      HT-1080 cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 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-LC3 Control Mutant displays a diffuse nuclear and cytosolic distribution in both fed and starved autophagic cells (i.e., no translocation to a punctate cytoplasmic distribution as characteristic of wild-type LC3).

      Immunocytochemistry Comparison and Inhibitor Analysis:
      (See Figure 2 in datasheet)
      Similar to Figure 1 (see datasheet), HeLa cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were either left in complete media, incubated for 4 hours in EBSS containing a lysosome inhibitor to induce autophagy and inhibit lysosomal degradation, or incubated as in, with the addition of 5 mM 3-methyladenine (3-MA) as an inhibitor of autophagy. 3-MA does not affect RFP-LC3 Control Mutant localization. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against LC3A reveals a similar expression pattern to the mutant protein (red) under fed conditions. Immunostaining of starved cells displays the punctate distribution of endogenous wild-type LC3, while signal following 3-MA treatment is diminished.

      Hard-to-transfect Cell Types:
      (See Figure 3 in datasheet)
      Primary cell types HUVEC (top row) or HuMSC (bottom row) were plated in chamber slides 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).

      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
      • MAP1LC3
      • LC3
      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
      Global Trade Item Number
      Catalog Number GTIN
      17-10188 04053252549236

      Documentation

      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor SDS

      Title

      Safety Data Sheet (SDS) 

      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor Certificates of Analysis

      TitleLot Number
      LentiBrite RFP-LC3 Control Mutant Lentiviral Biosensor - 2055469 2055469
      LentiBrite RFP-LC3 Control Mutant Lentiviral Biosensor - 2201532 2201532
      LentiBrite RFP-LC3 Control Mutant Lentiviral Biosensor - 2224455 2224455
      LentiBrite RFP-LC3 Control Mutant Lentiviral Biosensor - Q1988423 Q1988423
      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor - 2475931 2475931
      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor - 3560020 3560020
      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor - 3988510 3988510
      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor Packaged Lentiviral Particles 3136397
      LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor Packaged Lentiviral Particles 3047047

      Brochure

      Title
      Hallmarks of Aging

      Posters

      Title
      Poster: Autophagy Signaling