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17-10146 LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor

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Key Applications: Transfection, Immunofluorescence, Immunocytochemistry
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17-10146
1 vial (minimum of 3 x 10E8 IFU/mL)  
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      Descripción

      Replacement Information

      Tabla espec. clave

      Key ApplicationsDetection Methods
      TFX, IF, ICCFluorescent
      Description
      Catalogue Number17-10146
      Trade Name
      • LentiBrite
      • Chemicon
      DescriptionLentiBrite™ Calreticulin-RFP-KDEL 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™ Calreticulin-RFP-KDEL lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types.
      Alternate Names
      • Endoplasmic reticulum resident protein 60
      • Calregulin
      Background InformationCalreticulin is a resident protein of the lumen of the endoplasmic reticulum that plays important roles in protein folding and calcium homeostasis. In addition, cells induced to undergo apoptosis with selected agents allow passage of calreticulin from the ER to the cell surface, and the cell surface-exposed calreticulin promotes engulfment by phagocytic cells, a process termed immunogenic cell death. Expression of genetic fusions between calreticulin and fluorescent proteins has permitted analysis of the dynamics of chaperone-substrate interactions and immunogenic cell death.
      EMD Millipore’s LentiBrite™ Calreticulin-RFP-KDEL lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of calreticulin dynamics in difficult-to-transfect cell types.
      References
      Product Information
      Components
      • Calreticulin-TagRFP-KDEL 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 product box above.
      • 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), while others may require higher MOIs (e.g., human mesenchymal stem cells (HuMSC), 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
      Quality LevelMQ100
      Applications
      Key Applications
      • Transfection
      • Immunofluorescence
      • Immunocytochemistry
      Application NotesFluorescence Microscopy Imaging:
      (See Figure 1 in datasheet)
      U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 5 for 24 hours. After media replacement and 48 hours further incubation, cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluorescence microscopy.
      The calreticulin-RFP-KDEL appears in a meshwork pattern in the cytoplasm.

      Immunocytochemistry Comparison and Inhibitor Analysis:
      (See Figure 2 in datasheet)
      Similar to Figure 1, HeLa 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 48 hours. Immunocytochemical staining (green) of the same fields of view with a polyclonal antibody against calreticulin reveals similar expression patterns to the RFP-protein (red).

      Hard-to-transfect Cell Types:
      (See Figure 3 in datasheet)
      Primary cell types HUVEC and HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 and 10 respectively for 24 hours.

      Additional Cell Type:
      (See Figure 4 in datasheet)
      HT1080 and REF52 cells were treated as in Figure 1.

      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
      • CRP55
      • ERp60
      • HACBP
      • Grp60
      • CALR
      • CRTC
      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 target localization and 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 Merck 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
      Número de referencia GTIN
      17-10146 04053252639999

      Documentation

      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor Ficha datos de seguridad (MSDS)

      Título

      Ficha técnica de seguridad del material (MSDS) 

      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor Certificados de análisis

      CargoNúmero de lote
      LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor - N1956534 N1956534
      LentiBrite™ Calreticulin-RFP-KDEL -2763837 2763837
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 3321010 3321010
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 3598986 3598986
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 3722047 3722047
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 3989489 3989489
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 4028549 4028549
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor Packaged Lentiviral Particles 3134713
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 2458010 2458010
      LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor - 2282198 2282198

      Licencias necesarias e Información técnica

      Cargo
      LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation