17-10147
LentiBrite GFP-HMGB1 Lentiviral Biosensor
Synonym(s):
High mobility group protein B1, High mobility group protein 1
About This Item
Recommended Products
manufacturer/tradename
Chemicon®
LentiBrite
Quality Level
technique(s)
cell based assay: suitable
immunocytochemistry: suitable
immunofluorescence: suitable
transfection: suitable
UniProt accession no.
detection method
fluorometric
shipped in
dry ice
Gene Information
human ... HMGB1(3146)
General description
http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf
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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 GFP-HMGB1 lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of HMGB1 translocation in difficult-to-transfect cell types.
EMD Millipore’s LentiBrite GFP-HMGB1 lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of HMGB1 translocation in difficult-to-transfect cell types.
Application
(See Figure 1 in datasheet)
U2OS cells were plated in a chamber slide and transduced with lentiviral particles at an MOI of 10 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 GFP-HMGB1 displays in the nucleus of cells.
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 10 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. Immunocytochemical staining (blue) of the same fields of view with a nuclear stain (DAPI) reveals similar expression patterns to the protein-GFP (green).
Difficult-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC and Human mesenchymal stem cells (HuMSC) were plated in chamber slides and transduced with lentiviral particles at an MOI of 20 for 24 hours.
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.
Apoptosis & Cancer
Apoptosis - Additional
Components
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), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), human mesenchymal stem cells (HuMSC)).
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
Physical form
Storage and Stability
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.
Legal Information
Storage Class Code
10 - Combustible liquids
WGK
WGK 2
Regulatory Listings
Regulatory Listings are mainly provided for chemical products. Only limited information can be provided here for non-chemical products. No entry means none of the components are listed. It is the user’s obligation to ensure the safe and legal use of the product.
JAN Code
17-10147:
Certificates of Analysis (COA)
Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.
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