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17-10205 Sigma-Aldrich

LentiBrite™ RFP-Tubulin Lentiviral Biosensor

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Properties

trade name   LentiBrite
  Chemicon
detection method   Fluorescent
application(s)   Transfection
  Immunofluorescence
  Immunocytochemistry
  Track single cell and cell population migration (migration assay, cell culture)
purification method   PEG precipitation
gene symbol   TUB(7275)
UniProt accession no.   P07437
material size   1 vial (minimum of 3 x 10E8 IFU/mL)
shipped in   dry ice
storage conditions   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.

Description

Other Notes

See a time-lapse video of live cells undergoing tubulin bundling using the LentiBrite RFP-Tubulin Lentiviral Biosensor! Click Here

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 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-α-tubulin lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of microtubule dynamics in difficult-to-transfect cell types.

Background information

Microtubules are dynamic cytoskeletal filaments composed of tubulin subunits that play central roles during mitosis (in the mitotic spindle) and during interphase as a scaffold for directed kinesin- and dynein-mediated movement of cellular cargo. Several families of microtubule-binding agents, such as taxanes and vinca alkaloids, disrupt microtubule dynamics and cause cell death, and are clinically effective chemotherapeutic agents. Imaging of microtubule dynamics in live cells expressing tubulin tagged with fluorescent proteins has contributed greatly to our understanding of microtubule-binding agents.
EMD Millipore’s LentiBrite™ RFP-α-tubulin lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of microtubule dynamics in difficult-to-transfect cell types.

Components

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

Application

Fluorescence microscopy imaging:
HeLa cells 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 fixed with formaldehyde and mounted. Image was obtained by oil immersion confocal fluorescence microscopy. The RFP-tubulin displays predominantly cytoplasmic, fibrillar signal.

Immunocytochemistry Comparison and Modulator 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 20 for 24 hours. After media replacement and 48 hours further incubation, cells were either left untreated, incubated for 4 hours with 1 µM paclitaxel (PTX, a microtubule stabilizer), or incubated for 4 hours with 25 µM nocodazole (NZL, a microtubule depolymerization agent). PTX-treated cells become rounded and display the formation of tubulin “bundles”, while NZL eliminates fibrillar tubulin structure. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against α-tubulin reveals similar expression patterns to the RFP-protein (red).

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell types HUVEC or HuMSC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 for 24 hours. Cells were either left untreated or treated for 4 hours with 1 µM paclitaxel or 25 µM nocodazole.

Time-lapse Imaging:
(See Figure 4 in datasheet and video online)
HT-1080 cells were lentivirally transduced as in Figure 1. Shortly following addition of paclitaxel to a final concentration of 1 µM, time-lapse imaging was performed under temperature-controlled oil immersion wide-field fluorescence microscopy, with images taken every 1 minute over the course of 2 hours. Images demonstrate cellular rounding, microtubule organizing center migration, and the formation of stabilized tubulin “bundles”.

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.

Quality Assurance

Evaluated by transduction of HT-1080 cells and fluorescent imaging performed for assessment of 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 {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

Live Cell Imaging with Fluorescent Lentiviral Biosensors

Live Cell Imaging of β-Actin Cytoskeleton Proteins using LentiBrite™ Fluorescent Biosensors
Keywords: Apoptosis, Autophagy, Fluorescent microscopy, Gene expression, Microscopy, Transfection

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