Description
Key facts
Detection method:Fluorescent,
Sample types:Suspension cells, Adherent cells,
Assay type:Cell-based,
Assay time:30m,
Assay Platform:Microplate reader, Fluor. microscope, Flow cyt.
Product details:
Autophagy Assay Kit ab139484 offers a rapid and quantitative approach to monitoring autophagy in live cells without the need for cell transfection.It can be analyzed using flow cytometry, fluorescent microscopy, or using a microplate reader. The dye has spectral characteristics similar to FITC, and can be read with FITC channels / filters.
How the assay works
The Green Detection Reagent dye used in the Autophagy assay kit protocol has been optimized by screening dyes for both minimal staining of lysosomes, and bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes).
Autophagy inducer rapamycin is included in the kit as a positive control. Please note that the optimal final concentration is cell-dependent and should be determined experimentally for each cell line being tested. The agent has been validated in HeLa, HepG2 and Jurkat cells.
Chloroquine is included in the kit to use as an inhibitor control. Chloroquine may be used in combination with rapamycin or starvation in monitoring autophagic flux. Depending on the applications and specific cell lines, a 10-120 μM final concentration is recommended in order to observe changes in autophagic flux.
Autophagy assay protocol summary
- remove growth medium from cells
- add staining mix and incubate for 30 min
- wash cells
- analyze with fluorescence microscopy, flow cytometry, or fluorescent microplate reader
The reagents provided in this kit are sufficient for 200 flow cytometry tests, 250 fluorescence microscopy test or 3 x 96-well microplate tests.
How other researchers are using ab139484
Autophagy Assay Kit has been used in a variety of sample types including:
- Human normal hepatocytes and Human HCC cell lines 1
- Retinal endothelial cells, isolated from nondiabetic human retina 2
- 3T3-L1-adipocytes 3
References: 1 - Rajan P et al. 2023; 2 - Kowluru R et al. 2023; 3 - Park J at al. 2024.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Autophagy also known as "self-eating" is a cellular degradation and recycling process critical for maintaining cellular homeostasis. Mechanically autophagy involves the formation of double-membrane vesicles called autophagosomes which engulf damaged organelles and proteins. The autophagosomes then fuse with lysosomes leading to the degradation of the contents by lysosomal enzymes. Autophagy is expressed highly in cells under stress such as nutrient deprivation and is a conserved process across eukaryotic cells. Its machinery involves more than 30 autophagy-related genes (ATGs) but does not focus on a single mass or protein as it is a complex pathway.
Biological function summary
Autophagy protects cells by degrading and recycling components therefore preventing accumulation of damaged proteins and organelles. It forms part of the cellular defense mechanisms against stress and aging contributing to cellular longevity. In starvation conditions autophagy provides an internal source of nutrients helping cell survival. The process is part of a larger complex involving ATG proteins which drive the sequential steps of autophagosome formation. Monodansylcadaverine an autofluorescent compound often marks autophagic vacuoles in experimental settings providing a tool for autophagy detection and study.
Pathways
Autophagy is deeply integrated into cellular signaling networks. It plays a significant role in the mTOR (mechanistic target of rapamycin) signaling pathway which senses nutrient availability and regulates cell growth. Autophagy also intersects with the AMPK (AMP-activated protein kinase) pathway which responds to energy stress promoting catabolism when cellular ATP levels drop. These intersections with mTOR and AMPK pathways illustrate autophagy's essential role in balancing anabolic and catabolic processes and its regulatory association with proteins involved in cellular stress responses like p62/SQSTM1.
Autophagy is relevant to conditions like cancer and neurodegeneration. In cancer autophagy can have dual roles both supporting tumor cell survival under metabolic stress and limiting unregulated cell division. The Bcl-2 protein family which regulates apoptosis also modulates autophagy highlighting a complex interaction between cell death and survival. In neurodegenerative diseases such as Alzheimer's impaired autophagy leads to the accumulation of protein aggregates contributing to neuronal damage. Here proteins linked to autophagic dysfunction include beta-amyloid and tau both of which are involved in Alzheimer's disease pathology.
