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17-10196 Sigma-Aldrich

LentiBrite α-actinin-RFP Lentiviral Biosensor

  •  eCl@ss 34360190

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Properties

Quality Level   100
mfr. no.   Chemicon®
  LentiBrite
application(s)   cell analysis: suitable (cell based assay)
  immunocytochemistry: suitable
  immunofluorescence: suitable
  transfection: suitable
UniProt accession no.   P12814
detection method   fluorescent
shipped in   dry ice
Gene Information   human ... ACTN1(87)

Description

General description

Read our application note in Nature Methods!
http://www.nature.com/app_notes/nmeth/2012/121007/pdf/an8620.pdf
(_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 α-actinin-RFP lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types.

Migration of cells requires engagement of sites of extracellular matrix contact (focal adhesions) with the actin cytoskeleton. Contractile force is further generated when the actin microfilaments develop into stress fibers by bundling in an anti-parallel fashion and recruiting myosin II. A key component of stress fibers is the spectrin-related protein, α-actinin, which directly links actin microfilaments and also couples them to focal adhesions. Imaging of live cells containing α-actinin fused with fluorescent proteins has provided key insights into the dynamic mechanisms of stress fiber assembly and adaptation to strain.
EMD Millipore’s LentiBrite α-actinin-RFP lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of focal adhesion dynamics 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 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 α-actinin-RFP appears in a periodic distribution along microfilamentous structures.

Additional Cell Types:
(See Figure 2 in datasheet)
U2OS, HT1080 and REF52 cells were treated as in Figure 1 at an MOI of 5, 20 and 20, respectively, for 24 hours.

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell type HUVEC 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.

Research Category
Cell Structure

Research Sub Category
Adhesion (CAMs)

Components

α-actinin-TagRFP 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, human mesenchymal stem cells (HuMSC), human umbilical vein endothelial cells (HUVEC)), while others may require higher MOIs (e.g., HeLa, 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.

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 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.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

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 {HCompany} 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.

Safety & Documentation

Safety Information

Safety Information for this product is unavailable at this time.
Protocols & Articles

Articles

LentiBrite™ for fluorescent cell imaging for Cytoskeleton Structure

Cell analysis of the dynamics of subcellular structures has been revolutionized in the past 15 years by the development and refinement of genetically-encoded fusions between fluorescent proteins (GFP...
Keywords: Apoptosis, Cloning, Fluorescent microscopy, Gene expression, Immunocytochemistry, Immunofluorescence, Microscopy, Rearrangements, Tagged proteins, Transduction, Transfection

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