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17-10224 Sigma-AldrichLentiBrite™ GFP-p62 Lentiviral Biosensor

LentiBrite™ GFP-p62 Lentiviral Biosensor MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

SDS
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Key Applications: Transfection, Immunofluorescence, Immunocytochemistry

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Overview

Replacement Information

Key Specifications Table

Key Applications	Detection Methods
TFX, IF, ICC	Fluorescent

Description
Catalogue Number	17-10224
Trade Name	LentiBrite Chemicon
Description	LentiBrite™ GFP-p62 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™ GFP-p62 lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of autophagy in difficult-to-transfect cell types.
Alternate Names	Sequestosome-1 Sequestosome Ubiquitin-binding protein p62 EBI3-associated protein p62
Background Information	Autophagy, a degradative pathway induced in cells under stress, plays both protective and deleterious roles in many diseases, including cancer, neurodegeneration, and infections. The adaptor protein p62 targets protein aggregates to the autophagosome for degradation via its polyubiquitin-binding domain, and also functions as a scaffold for signaling proteins such as atypical PKCs and mTORC1. In many cell types, p62 ordinarily exists as puncta or speckles, which increase in number and size upon blockade of autophagic flux with lysosome inhibitors. Upon initiation of autophagy, the p62-cargo protein complex binds to LC3 on the autophagosome surface, which results in degradation of this complex. DNA constructs encoding fluorescent proteins fused to p62 are often introduced into cells for monitoring aggregate formation by fluorescence microscopy. EMD Millipore’s LentiBrite™ GFP-p62 lentiviral particles provide bright fluorescence and precise localization to enable live-cell analysis of autophagy in difficult-to-transfect cell types.

References

Product Information
Components	TagGFP2-p62 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, HeLa, U2OS, human mesenchymal stem cells (HuMSC)), while others may require higher MOIs (e.g., human umbilical vein endothelial cells (HUVEC)). 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
Application Notes	Fluorescence Microscopy Imaging: (See Figure 1 in datasheet) Primary human mesenchymal stem cells (HuMSC) were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 24 hours further incubation, cells were either left in complete media or incubated for 4 hours in EBSS containing a lysosome inhibitor, to induce autophagsome formation and inhibit lysosomal degradation. Cells were fixed with formaldehyde, mounted and imaged by oil immersion wide-field fluorescence microscopy. The GFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells. Immunocytochemistry Comparison: (See Figure 2 in datasheet) Similar to Figure 1 (see datasheet), U2OS cells were plated in a chamber slide and transduced with GFP-p62 lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 24 hours. The cells were then fixed and stained with a polyclonal antibody against p62 (Cat. No. MABC32), followed by a Cy3-conjugated anti-rabbit IgG. Distribution of the GFP-p62 (green) is similar to that detected by immunocytochemical staining (red). Hard-to-transfect Cell Types: (See Figure 3 in datasheet) Primary cell type HUVEC were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 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 (see datasheet) Time-lapse Imaging: (See Figure 5 in datasheet and video online) U2OS cells were plated in coverglass chamber slides and transduced with GFP-p62 lentiviral particles as in Figure 1 (see datasheet). Images were collected every 20 seconds for a total of 32 min. Shown here are 3 sequential frames. The GFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells. 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

Application Notes

Fluorescence Microscopy
Imaging:
(See Figure 1 in datasheet)
Primary human mesenchymal stem cells (HuMSC) were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 24 hours further incubation, cells were either left in complete media or incubated for 4 hours in EBSS containing a lysosome inhibitor, to induce autophagsome formation and inhibit lysosomal degradation. Cells were fixed with formaldehyde, mounted and imaged by oil immersion wide-field fluorescence microscopy.
The GFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

Immunocytochemistry Comparison:
(See Figure 2 in datasheet)
Similar to Figure 1 (see datasheet), U2OS cells were plated in a chamber slide and transduced with GFP-p62 lentiviral particles at an MOI of 40 for 24 hours. After 24 hours, media was replaced and cells were then further incubated for 24 hours. The cells were then fixed and stained with a polyclonal antibody against p62 (Cat. No. MABC32), followed by a Cy3-conjugated anti-rabbit IgG. Distribution of the GFP-p62 (green) is similar to that detected by immunocytochemical staining (red).

Hard-to-transfect Cell Types:
(See Figure 3 in datasheet)
Primary cell type HUVEC were plated in a chamber slide and transduced with lentiviral particles at an MOI of 40 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 (see datasheet)

Time-lapse Imaging:
(See Figure 5 in datasheet and video online)
U2OS cells were plated in coverglass chamber slides and transduced with GFP-p62 lentiviral particles as in Figure 1 (see datasheet). Images were collected every 20 seconds for a total of 32 min. Shown here are 3 sequential frames. The GFP-p62 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

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	p62 SQSTM1 ORCA OSIL
Purification Method	PEG precipitation
UniProt Number	Q13501

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 MilliporeSigma 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 MilliporeSigma 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
Catalog Number	GTIN
17-10224	04053252819216

Documentation

LentiBrite™ GFP-p62 Lentiviral Biosensor SDS

Title
Safety Data Sheet (SDS)

LentiBrite™ GFP-p62 Lentiviral Biosensor Certificates of Analysis

Title	Lot Number
LentiBrite GFP-p62 Lentiviral Biosensor - Q2064881	Q2064881
LentiBrite GFP-p62 -2526212	2526212
LentiBrite GFP-p62 -2556372	2556372
LentiBrite GFP-p62 -2760511	2760511
LentiBrite GFP-p62 Lentiviral Biosensor - 2998370	2998370
LentiBrite GFP-p62 Lentiviral Biosensor - 3305349	3305349
LentiBrite GFP-p62 Lentiviral Biosensor - 3481376	3481376
LentiBrite GFP-p62 Lentiviral Biosensor - 3767424	3767424
LentiBrite GFP-p62 Lentiviral Biosensor - 3855699	3855699
LentiBrite GFP-p62 Lentiviral Biosensor - 3918060	3918060

Brochure

Title
Hallmarks of Aging

Posters

Title
Poster: Autophagy Signaling

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17-10224 Sigma-AldrichLentiBrite™ GFP-p62 Lentiviral Biosensor

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Overview

Key Specifications Table

Documentation

LentiBrite™ GFP-p62 Lentiviral Biosensor SDS

LentiBrite™ GFP-p62 Lentiviral Biosensor Certificates of Analysis

Brochure

Posters

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