LentiBrite Calreticulin-RFP-KDEL Lentiviral Biosensor

Description

• General description: Read 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 HereBiosensors 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.
• Application: Fluorescence 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.
• 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. PromoterEF-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.
• Quality: Evaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of target localization and transduction efficiency.
• Physical form: PEG precipitation
• Storage and Stability: Storage and HandlingLentivirus 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 NOTEReplication-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.
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