FCCH100183 FlowCellect™ RFP-LC3 Reporter Autophagy Assay Kit

FCCH100183
100 Tests  
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      Overview

      Replacement Information
      Description
      Catalogue NumberFCCH100183
      Trade Name
      • FlowCellect
      DescriptionFlowCellect™ RFP-LC3 Reporter Autophagy Assay Kit
      OverviewMillipore’s FlowCellect™ RFP-LC3 Reporter Autophagy Assay Kit provides a quantitative solution for the study of autophagy and potency evaluation of autophagy inducers using flow cytometry. This kit has four unique features:

      1) The use of selective permeabilization solution discriminates cytosolic LC3 from autophagic LC3 by extracting the soluble cytosolic proteins, while protecting LC3 which has been sequestered into the autophagosome from degradation;
      2) A monomeric RFP is used as a reporter to facilitate the translocation of the fusion protein. Other forms of RFP are not preferred as they form dimers and aggregate when over-expressed in the cells, preventing their extraction from the cytoplasm and impairing the ability to measure translocation by flow cytometry;
      3) Since autophagy is a constitutive cellular degradation process, the use of an autophagy detection reagent (Autophagy Reagent A) will prevent the lysosomal degradation of LC3, allowing its quantification by flow cytometry;
      4) The monomeric RFP used in our fusion protein to LC3 is attached on the 5' end (N-terminal fusion), protecting the RFP from Atg4 cleavage, thus, allowing its visualization within the autophagosomes (figure 3).

      To assist in autophagy study, U20S (human osteosarcoma) cells are stably transfected, which are suitable for both imaging and flow cytometry instruments.
      Alternate Names
      • FlowCellect
      Background InformationAutophagy is an intracellular catabolic pathway that causes cellular protein and organelle turnover, and it is associated in diverse diseases including Alzheimer's disease, aging, cancers, and Crohn's disease. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism by disposing of aging and/or dysfunctional proteins and organelles by sequestering and priming for lysosomal degradation. Increasing evidence suggests that not just apoptosis, but autophagy can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can influence longevity and productivity of cells to function at full capacity.

      During autophagy, LC3 protein is translocated from the cytoplasm to the autophagosome where it is targeted to the lysosome for degradation. The process of autophagy can be categorized into four distinct stages:

      1) Induction and LC3 Translocation: The process is initiated by external/internal stimuli (e.g. nutrient depletion);
      2) Autophagosome formation: Unwanted cytosolic proteins and aging organelles are sequestered by a double membrane vesicle, i.e.—”autophagosome”. Formation of this vesicle is coordinated by complexes of Atg proteins (Atg5 and Atg12) that are conjugated, enabling the recruitment of LC3;
      3) Lysosomal docking and fusion: LC3 protein regulates traffic between autophagosome to lysosome. (LC3-I is cytoplasmic; LC3-II is lipidated and sequestered into autophagosomal membrane);
      4) Degradation: Fusion with the lysosome and subsequent breakdown of the autophagic vesicle and its contents.
      References
      Product Information
      Components
      • 1. U20S cell background (Part No. CF200094) One vial containing 5 million cells
      • 2. Autophagy reagent A: (Part No. CS208212) One vial (lyophilized)
      • 3. Autophagy reagent B: (Part No. CS208215) One vial containing 1 mL
      • 4. 5X Assay Buffer: (Part No. CS202124) One bottle containing 55 mL
      Detection methodFluorescent
      FormatCell Line
      Instrument compatibility
      • EasyCyte 8HT
      • EasyCyte
      Applications
      ApplicationThis FlowCellect RFP-LC3 Reporter Autophagy Assay Kit for Flow Cytometry provides a quantitative solution for the study of autophagy & potency evaluation of autophagy inducers using flow cytometry.
      Key Applications
      • Flow Cytometry
      Biological Information
      Species Reactivity
      • Human
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • 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 ConditionsThis is a dual storage kit. All reagents must be stored at 2 - 8ºC, and the cell line must be stored at either -80°C (short term) or in liquid nitrogen (long term).
      Packaging Information
      Material Size100 Tests
      Transport Information
      Supplemental Information
      Specifications

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      Categories

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