Autophagy Assay

by Promega
Available SKUs: GA1040 | GA1050 | GA2550 |
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Description

  • Overview
  • Protocols
  • Specifications
  • Resources
  • Simple Bioluminescent Detection of Autophagic Flux

    The Autophagy LC3 HiBiT Reporter Assay System is a plate-based assay using a luminescent LC3 reporter to quantitatively measure autophagic flux. The easy one-step protocol eliminates wash steps and the need for complex imaging or flow cytometry systems. Choose from one of two prepared reporter cell lines (HEK293 or U2OS) or stably transfect the reporter vector into the cell line of your choice.

    How the Assay Works

    The Autophagy LC3 HiBiT Reporter consists of a HiBiT tag fused to the human LC3 protein. When autophagy is induced, LC3 reporter proteins are captured in autophagosomes and degraded. With addition of the Nano-Glo® HiBiT Lytic Reagent containing LgBiT protein and substrate, LgBiT interacts with the HiBiT tag to reconstitute the bright, luminescent NanoBiT® enzyme. The luminescent signal is directly proportional to the level of HiBiT-tagged LC3 reporter over seven orders of magnitude.

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    What The Data Looks Like

    The readout from the autophagy reporter assay is quantitative and easy to interpret. Increased autophagic flux will accelerate the degradation of the autophagy reporter, resulting in decreased luminescent signal. In contrast, inhibition of autophagy will increase reporter levels and luminescent signal.

     

    Autophagy Induction: Decreased Signal

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    HEK293 Autophagy LC3 HiBiT Reporter cells were plated into all-white, 96-well plates at 20,000 cells/well. After cells attached overnight, treatment was performed in triplicate for the indicated times with increasing concentrations of a reference autophagy inducer, PP242. Nano-Glo® HiBiT Lytic Reagent was then added and luminescence measured after 10 minutes. Average signal is reported as normalized luminescence relative to time-matched, vehicle-treated controls.

    Autophagy inhibition: Increased Signal

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    U2OS Autophagy LC3 HiBiT Reporter Cells were plated into all-white, 96-well plates at 8,000 cells/well.  After cells attached overnight, cells were treated with increasing concentrations of a reference inhibitor, Baf A1, without or with 2μM PP242 for 21 hours. Luminescent signal was normalized to untreated control for each study. Treatment with PP242 significantly increased autophagy and decreased luminescent signal to allow more capacity for signal increase by cotreatment with the autophagy inhibitor, Baf A1.

    See More Data: Learn more about this data and see how the Autophagy LC3 HiBiT Reporter Assay can be used with 3D cell models in the poster Assessing Autophagic Flux in 2D and 3D Cell Culture Models with a Novel Plate-Based Assay, presented at ASCB 2017. 

    Autophagy LC3 HiBiT Reporter Vector

    The Autophagy LC3 HiBiT Reporter consists of human LC3B, an N-terminal HiBiT tag, and an intervening “Spacer” region that enhances reporter specificity for the autophagic pathway. It utilizes the constitutive HSV-TK promoter with PyF101 enhancer for low-to-moderate expression of the reporter in mammalian cells. The vector also includes a Neo-Kan resistance gene, allowing generation of stable clonal lines via selective pressure with Geneticin (G418).

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    Autophagy LC3 HiBiT Reporter Vector sequence text file.
  • Specifications

    What's in the box?

    Item Part # Size

    Nano-Glo® HiBiT Lytic Detection System

    N3030 1 × 10ml

    HEK293 Autophagy LC3 HiBiT Reporter Cell Line

    GA104A 1 × 1 each

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.
  • Patents and Disclaimers

    NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If the researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

    This product may not be further sold or transferred by the researcher and may be used only by the researcher, and then only for research use, which includes, but is not limited to drug discovery and development. No other commercial use is allowed. "Commercial use" means any and all uses of this product by researcher for monetary or other consideration, including, but not limited to, (1) use in further product manufacture; (2) use in provision of services, information or data to unaffiliated third parties, and (3) resale of this product for any use. Researcher has no right to modify, derivatize, genetically engineer or otherwise create variations of the nucleotide sequence encoding the HiBiT and HaloTag® peptides which sequences have been stably transfected within the cells. Researcher may genetically engineer the cell line using exogenous nucleic acid sequences of the researcher’s choosing and researcher may propagate and store the cells for long-term use. In addition, researcher must use Nano-Glo® HiBiT Lytic Detection System purchased from Promega for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. For uses of HaloTag® Technology in this product, researchers must either: (1) use Promega HaloTag® ligands, which can be modified or linked to Promega or customer-supplied moieties, or (2) contact Promega to obtain a license if Promega HaloTag® ligands are not to be used.

    With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

    BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.

    Researcher may use this product for research use only; no commercial use is allowed. Commercial use means any and all uses of this product by a party in exchange for consideration, including, but not limited to (1) use in further product manufacture; (2) use in provision of services, information or data; and (3) resale of the product, whether or not such product is resold for use in research. Researcher shall have no right to modify or otherwise create variations of the product. No other use or transfer of this product is authorized without the prior express written consent of Promega.

    For uses of Nano-Glo®-branded reagents intended for energy transfer (such as bioluminescence resonance energy transfer) to acceptors other than a genetically encoded autofluorescent protein, researchers must:
    (a) use NanoBRET™-branded energy acceptors (e.g., BRET-optimized HaloTag® ligands) for all determinations of energy transfer activity by this product; or
    (b) contact Promega to obtain a license for use of the product for energy transfer assays to energy acceptors not manufactured by Promega.

    With respect to any uses outside this label license, including any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WITH REGARD TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

    U.S. Pat. No. 9,797,890 and other patents pending.

    Licensed from Kazusa DNA Research Institute.

    Licensed under U.S. Pat. Nos. 6,632,672, 7,361,641 and 8,227,249.

    HEK293 cells were obtained under license from AdVec Inc.

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