FlowCellect(R）Autophagy LC3 Antibody-based Assay Kit

The Guava^(R) Autophagy LC3 Antibody-based Detection Kit ー100 Tests (Part Number: FCCH100171) provides a quantitative solution to study autophagy and evaluate autophagy inducers using flow cytometry. This kit contains two key detection reagents to facilitate the monitoring of lipidated LC3-II in a given cell system:

1. A 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

2. An autophagy detection reagent (Autophagy Reagent A), which will prevent the lysosomal degradation of LC3, allowing for its quantification by flow cytometry

Autophagy is an intracellular catabolic pathway that causes cellular protein and organelle turnover and is associated with a range of diseases. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy is a housekeeping mechanism that works by disposing of aging and dysfunctional proteins and organelles through sequestering, and by priming proteins for lysosomal degradation. Malfunctions in the autophagy process are proposed to influence cell health longevity as well as contribute to cell death.

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

1. Induction and LC3 translocation: 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., the autophagosome); formation of this vesicle is coordinated by complexes of conjugated Atg proteins (Atg5 and Atg12), enabling the recruitment of LC3

3. Lysosomal docking and fusion: The LC3 protein regulates traffic between the autophagosome to the lysosome (LC3-I is cytoplasmic; LC3-II is lipidated and sequestered into autophagosomal membrane)

4. Degradation: Fusion with the lysosome and the subsequent breakdown of the autophagic vesicle and its contents

By measuring and quantifying autophagy, we are able to screen and rank order autophagy inducers and inhibitors, monitor cell culture health and protein turnover rate, study the mechanisms of protein degradation, and identity new autophagy targets and pathways leading to aging and neurodegenerative diseases.

In this kit, an anti-LC3 antibody is conjugated to FITC; this and the accompanying autophagy-enabling reagents have been carefully evaluated to ensure optimal performance, alleviating the need for any additional validation of the kit reagents. The reagents provide a complete solution for autophagy analysis.

Components:20X Anti-LC3 FITC, clone 4E12, Autophagy Reagent A, Autophagy Reagent B, 5X Assay Buffer

Detection Method:Fluorescence

For Research Use Only. Not for use in diagnostic procedures.