Description
Key facts
Sample types:Adherent cells,
Assay type:Quantitative,
Assay Platform:Fluorescence microscope
Product details:
Autophagy/Cytotoxicity Dual Staining Kit (ab133075) provides a convenient tool for studying the regulation of autophagy and cytotoxicity at the cellular level. The kit employs monodansylcadaverine (MDC), a fluorescent compound that is incorporated into multilamellar bodies by both an ion trapping mechanism and the interaction with membrane lipids, as a probe for detection of autophagic vacuoles in cultured cells. Propidium iodide (PI) is used as a marker of cell death. Tamoxifen, a known inducer of autophagy, is included as a positive control. This kit provides sufficient reagent to effectively treat/stain 960 individual wells of cells when utilized in a 96-well plate format. Lower density plates will still require approximately the same amount of reagent on a per plate basis. Therefore, up to 10 plates worth of cells can be examined irrespective of the number of wells/plate (this is not the case for protocols that use non-adherent cells).
Autophagy is a critical cellular process that involves the degradation and digestion of intracellular components by the lysosome. This process not only enables cells to efficiently mobilize and recycle cellular constituents, but also prevents the accumulation of damaged organelles, misfolded proteins, and invading microorganisms.
Autophagy is a multi-step process that begins with the sequestration of cytoplasmic organelles and proteins. These cellular components are sequestered by a double membrane, forming an autophagosome. The autophagosome then fuses with a lysosome to form an autolysosome, where the cellular material is then degraded. Normal autophagy is essential for survival, differentiation, development, and homeostasis. Dysregulation of autophagy has been implicated in cancer, infection, aging, and degenerative diseases.
While autophagy most often acts to promote cell survival in response to stress, it can also promote cell death. The relationship between autophagy and apoptosis is complex. The two pathways share common stimuli and components, and can regulate the activity of each other. However, the specific factors and mechanisms that dictate the choice between autophagy and apoptosis remain unclear.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
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.
