Description
General description
Also available: LentiBrite GFP-p62 Lentiviral Biosensor! Click Here
Also available: LentiBrite RFP-p62 Lentiviral Biosensor! Click Here
Read our application note in Nature Methods!
http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf
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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 lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types.
Autophagy, a degradative pathway that provides recycled nutrients to cells under stress, plays both protective and deleterious roles 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, diffusely distributed in the cytosol, are proteolytically processed to form LC3-I. Upon initiation of autophagy, the C-terminal glycine is converted to LC3-II by addition of a phosphatidylethanolamine by Atg5-Atg12. LC3-II translocates rapidly to nascent autophagosomes in a punctate distribution. DNA constructs encoding fluorescent proteins fused to LC3 are widely employed for introduction into cells for monitoring autophagosome formation by fluorescence microscopy.
EMD Millipore’s LentiBrite RFP-LC3 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)
Primary cell type, Human mesenchymal stem cells (HuMSC), 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 fluores-cence microscopy. The RFP-LC3 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.
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 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 completely blocks formation of RFP-LC3-positive autophagic punctae. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against LC3A reveals similar expression patterns to the RFP-protein (red).
Hard-to-transfect Cell Type:
(See Figure 3 in datasheet)
Primary cell type HUVEC were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 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.
Confocal Microscopy Imaging:
(See Figure 4 in datasheet)
HeLa cells were treated as in Figures 1A and 1B. Images were obtained by oil immersion confocal fluorescence microscopy.
Additional Cell Type:
(See Figure 5 in datasheet)
HT1080 cells were treated as in Figure 1A and 1B.
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.
Analysis Note
Evaluated by transduction of HT-1080 cells at MOI values of 20 and 40. Fluorescent imaging performed for assessment of target localization and transduction efficiency.
Other Notes
TagRFP-LC3 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), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), U2OS, 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.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
