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17-10222 Sigma-AldrichLentiBrite™ EB3-RFP Lentiviral Biosensor

LentiBrite™ EB3-RFP Lentiviral Biosensor: SDB (Sicherheitsdatenblätter), Analysenzertifikate und Qualitätszertifikate, Dossiers, Broschüren und andere verfügbare Dokumente.

SDB
Analysenzertifikat

Key Applications: Transfection, Immunofluorescence, Immunocytochemistry, Track single cell and cell population migration (migration assay, cell culture)

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17-10222

1 vial (minimum of 3 x 10E8 IFU/mL)

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Key Spec Table

Key Applications	Detection Methods
TFX, IF, ICC, Track single cell and cell population migration (migration assay, cell culture)	Fluorescent

Description
Catalogue Number	17-10222
Trade Name	LentiBrite Chemicon
Description	LentiBrite™ EB3-RFP Lentiviral Biosensor
Overview	*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™ EB3-RFP lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of microtubule plus-end dynamics in difficult-to-transfect cell types.
Background Information	Microtubules are dynamic cytoskeletal filaments comprising a core of tubulin subunits flanked by structurally and functionally distinct termini, termed the plus-end and minus-end. The dynamic instability of microtubules, with cycles of growth and decay, occurs at the plus-ends and is mediated by a complex of microtubule plus-end tracking proteins (+TIPs). Key components of the +TIP complex are the end-binding proteins (EBs). Imaging of plus-end growth in live cells expressing EBs tagged with fluorescent proteins has contributed greatly to our understanding of microtubule dynamics. EMD Millipore’s LentiBrite™ EB3-RFP lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of microtubule plus-end dynamics in difficult-to-transfect cell types.

References

Product Information
Components	EB3-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 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 mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC), U2OS, HeLa). 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.
Detection method	Fluorescent
Quality Level	MQ100

Applications
Key Applications	Transfection Immunofluorescence Immunocytochemistry Track single cell and cell population migration (migration assay, cell culture)
Application Notes	Fluorescence Microscopy Imaging: REF52 cells were plated in a coverglass 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 imaged live by oil immersion wide-field fluorescence microscopy. The EB3-RFP displays a punctate distribution characteristic of microtubule ends. Immunocytochemistry Comparison and Inhibitor Analysis: HeLa cells were plated in a coverglass chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. EB3-RFP displays a nearly complete overlap with EB3 detected by immunocytochemical staining with rabbit polyclonal anti-EB3 (EMD Millipore cat. no. AB6033). Additional Cell Types: MCF7 cells were transduced with EB3-RFP lentivirus as in Figure 1 at an MOI 40 for 24 hours, then stained for tubulin. EB3-RFP displays a punctate and short linear distribution. Immunocytochemical staining with a monoclonal antibody against α-tubulin (EMD Millipore Cat. No. 05-829) reveals the typical filamentous pattern of microtubules. Overlay of EB3-RFP (red), anti-tubulin (green) and DAPI (blue) demonstrates that EB3-RFP is localized to the microtubule ends. Hard-to-transfect Cell Types: Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in coverglass chamber slides and transduced with lentiviral particles at an MOI of 20 and 40, respectively, for 24 hours. Time-lapse Imaging: REF52 cells were plated in coverglass chamber slides and transduced with EB3-RFP lentiviral particles. Images were collected every 3 seconds for a total of 2 min. Shown here are 3 sequential frames; to see the entire sequence, please visit our Cell Structure-Biosensors page. 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.

Applications

Key Applications

Transfection
Immunofluorescence
Immunocytochemistry
Track single cell and cell population migration (migration assay, cell culture)

Application Notes

Fluorescence Microscopy Imaging:
REF52 cells were plated in a coverglass 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 imaged live by oil immersion wide-field fluorescence microscopy. The EB3-RFP displays a punctate distribution characteristic of microtubule ends.

Immunocytochemistry Comparison and Inhibitor Analysis:
HeLa cells were plated in a coverglass chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 48 hours. EB3-RFP displays a nearly complete overlap with EB3 detected by immunocytochemical staining with rabbit polyclonal anti-EB3 (EMD Millipore cat. no. AB6033).

Additional Cell Types:
MCF7 cells were transduced with EB3-RFP lentivirus as in Figure 1 at an MOI 40 for 24 hours, then stained for tubulin. EB3-RFP displays a punctate and short linear distribution. Immunocytochemical staining with a monoclonal antibody against α-tubulin (EMD Millipore Cat. No. 05-829) reveals the typical filamentous pattern of microtubules. Overlay of EB3-RFP (red), anti-tubulin (green) and DAPI (blue) demonstrates that EB3-RFP is localized to the microtubule ends.

Hard-to-transfect Cell Types:
Primary cell type HUVEC and Human mesenchymal stem cells (HuMSC) were plated in coverglass chamber slides and transduced with lentiviral particles at an MOI of 20 and 40, respectively, for 24 hours.

Time-lapse Imaging:
REF52 cells were plated in coverglass chamber slides and transduced with EB3-RFP lentiviral particles. Images were collected every 3 seconds for a total of 2 min. Shown here are 3 sequential frames; to see the entire sequence, please visit our Cell Structure-Biosensors page.

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.

Biological Information
Gene Symbol	MAPRE3
Purification Method	PEG precipitation
UniProt Number	Q9UPY8

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
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 Merck 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.

Product Usage Statements

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 Merck 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.

Storage and Shipping Information
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.

Storage and Shipping Information

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.

Packaging Information
Material Size	1 vial (minimum of 3 x 10E8 IFU/mL)

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Bestellnummer	GTIN
17-10222	04053252698040

Documentation

LentiBrite™ EB3-RFP Lentiviral Biosensor SDB

Titel
Sicherheitsdatenblatt (SDB)

LentiBrite™ EB3-RFP Lentiviral Biosensor Analysenzertifikate

Titel	Chargennummer
LentiBrite EB3-RFP Lentiviral Biosensor - Q2043551	Q2043551
LentiBrite EB3-RFP -2770272	2770272
LentiBrite EB3-RFP Lentiviral Biosensor - 3173614	3173614
LentiBrite EB3-RFP Lentiviral Biosensor - 3407273	3407273
LentiBrite EB3-RFP Lentiviral Biosensor - 3669291	3669291
LentiBrite EB3-RFP Lentiviral Biosensor - 3831828	3831828
LentiBrite EB3-RFP Lentiviral Biosensor - 4138107	4138107
LentiBrite EB3-RFP Lentiviral Biosensor Packaged Lentiviral Particles	2867483

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Kategorien

Life Science Research > Cell Analysis > Cell-based Assays > Lentiviral Biosensors

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