Fluorescent dyes can be used to stain tissue sections or cells to observe the occurrence of autophagy. In the late stage of autophagy, autophagosomes fuse with lysosomes to become acidic. According to this feature, autophagy lysosomes can be labeled with acid-tropic dyes Dansylcadaverine (MDC), Acridine Orange (AO), and LysoTracker.
Creative Bioarray has developed a series of autophagy research techniques. We can undertake various types of cell autophagy testing, and can provide a full set of customized technical services such as cell culture, cell processing, image collection, and data measurement. Our services include but are not limited to:
MDC Staining Service
MDCis an eosinophilic fluorescent complex and is the earliest dye used for autophagosome labeling. MDC can bind to autophagosomes. MDC staining can be used to observe whether the autophagy process has occurred. MDC stained cells with autophagic vesicles can be detected by flow cytometry or fluorescence microscopy. A microplate reader can also be used to directly detect the amount of MDC dye binding in cells. Studies have shown that the number of spots stained positive for MDC is related to autophagy activity. Under the excitation of ultraviolet light, the autophagic vesicles showed bright blue fluorescence.
However, the MDC method is not specific for detecting early autophagy, only for late autophagy in the material degradation stage. MDC not only binds to autophagosomes, but also binds to all acidic vacuoles in the cytoplasm. Therefore, it is recommended to combine different methods to detect early and late autophagy.
AO Staining Service
AO is an important weak alkaline stain for detecting acidic vesicle structure. AO is cell-permeable and can freely cross the membrane and stain DNA and cytoplasm bright green. AO can also penetrate acidic organelles (e.g., autophagic lysosomes). In a low pH environment, the red fluorescence emitted by AO is proportional to acidity. Cells stained with AO dye can be detected by flow cytometry or fluorescence microscopy. Under a fluorescence microscope, it can be seen that cells without autophagic lysosomes appear uniformly green, while cells with autophagic lysosomes appear red.
AO is suitable for the detection of autophagy lysosomes and their undegradable product residues such as lipofuscin particles. This method is best combined with other detection methods for a comprehensive evaluation.
LysoTracker Staining
LysoTracker is an acidic indicator that tends to accumulate in low pH cell environments. Lyso Tracker can be transported to the lysosome through endocytosis. When the pH of the lysosome is lower than 5, this indicator will form a strong fluorescence in the lysosome. It can assist the quantification of lysosomes in cells and detect the efficiency of autophagosome and lysosome fusion in cells.
These fluorescent dyes mentioned above can stain all acidic vacuoles, so not all stain-positive particles are autophagic lysosomes. Therefore, this method does not specifically reflect the activity of white phages, and must be used in conjunction with other autophagy detection methods to determine the activity of autophagy. Creative Bioarray provides you with a variety of autophagy detection methods and one-stop services that you can choose as needed.
The time depends on the experiment content
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