17-10230

LentiBrite GFP-LC3-II Enrichment Kit (Flow Cytometry)

Name: LentiBrite GFP-LC3-II Enrichment Kit (Flow Cytometry)
Brand: MM

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About This Item

UNSPSC Code:

12161503

eCl@ss:

32161000

NACRES:

NA.84

technique(s)

flow cytometry: suitable

Quality Level

100

detection method

fluorometric

General description

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 modified by addition of a phosphatidylethanolamine (PE) to form LC3-II, which 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. <br />EMD Millipore′s LentiBrite GFP-LC3 lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types. The <br /><br />LentiBrite GFP-LC3-II Enrichment Kit (Flow Cytometry) combines our GFP-LC3 lentiviral particles with a selective permeabilization procedure for GFP-LC3 transduced cells that allows release of cytosolic LC3-I and retention of autophagosome-bound LC3-II. This procedure allows detection of autophagosome density by flow cytometry.

Application

EMD Millipore′s LentiBrite GFP-LC3 lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of autophagy in difficult-to-transfect cell types. The LentiBrite GFP-LC3-II Enrichment Kit (Flow Cytometry) combines our GFP-LC3 lentiviral particles with a selective permeabilization procedure for GFP-LC3 transduced cells that allows release of cytosolic LC3-I and retention of autophagosome-bound LC3-II. This procedure allows detection of autophagosome density by flow cytometry.

For optimal fluorescent analysis, 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.

Preparation Note

Store TagGFP2-LC3 Lentivirus 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. Store Autophagy Reagent A, 10x Enrichment Buffer, and 5x Assay Buffer at 2-8°C. All components are stable for at least 4 months from date of receipt when stored as recommended above.

Other Notes

1. TagGFP2-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 datasheet. • Virus Description: 3rd generation HIV-based VSV-G pseudotyped lentiviral particles. Lentiviral particles were generated using the pPACKH1 Lentivector Packaging System and purified by PEG precipitation. • Promoter: EF-1 (Elongation Factor-1). • Expression Marker Description: TagGFP2 (Evrogen) fused at its C-terminus with human LC3A (Accession # NP_115903) truncated to leave G120 (lipidation site) at the C-terminus. (Store at -80°C) 2. Autophagy Reagent A: One vial (lyophilized) (Store at 2-8°C) 3. 10x Enrichment Buffer (Part No. CS210552): One vial containing 1 mL (Store at 2-8°C) 4. 5x Assay Buffer: (Part No. CS210529): One bottle containing 3 mL (Store at 2-8°C)

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.

Pictograms

GHS07

Signal Word

Warning

Hazard Statements

H302,H412

Precautionary Statements

P264 - P270 - P273 - P301 + P312 - P501

Hazard Classifications

Acute Tox. 4 Oral - Aquatic Chronic 3

Storage Class Code

10 - Combustible liquids

WGK

WGK 3

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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With EMD Millipore, epigenetics technology has never been more accessible or easier to use. From highly specific antibodies, to rapid bisulfite kits, to industry leading ChIP and next generation sequencing kits, we simplify epigenetics applications to allow you to design the most ambitious experiments to interrogate gene regulation. With our legacy of expertise from Chemicon® and Upstate®, we are committed to molding sophisticated yet simple solutions for understanding epigenetic regulation.

LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation

Expression of genetically-encoded fluorescently-tagged proteins has widely been employed for real-time visualization of cellular behavior and trafficking. Prepackaged, ready-to-use, high-titer lentiviral particles (which we have termed “lentiviral biosensors”) encoding GFP- or RFP-tagged proteins are a convenient, robust solution for fluorescent imaging of transduced cells. Compared to other nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are non-disruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 for the study of autophagosome formation.

Autophagy Signaling

Autophagy Signaling Pathways and Detection

Global Trade Item Number

SKU	GTIN
17-10230	04053252008795

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