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Cat. No.: AFAL-200 (for 20-200T)

Cat. No.: AFAL-1k (for 100-1000T)

Description

Autolysosome Fluorescence Staining Kit with AL-Green is a kit for rapid and convenient detection of autolysosomes in live cells using AL-Green as the fluorescent probe. This kit is compatible with fluorescence detection systems including fluorescence microscopes, laser scanning confocal microscopes, and flow cytometers.

Autophagy is an evolutionarily highly conserved intracellular catabolic pathway, which is achieved by lysosomes engulfing and degrading portions of cellular components. Autophagy is closely associated with multiple physiological functions. For instance, under stress conditions such as starvation, cells can degrade excess or abnormal intracellular components via autophagy to supply energy and raw materials for cell survival, thereby sustaining organismal growth and development, cell differentiation, and environmental adaptability. Once autophagy is initiated, cells form a double-membrane isolation membrane that gradually extends to enclose damaged cellular components (such as proteins or organelles), ultimately generating autophagosomes. Autophagosomes then fuse with lysosomes to form autolysosomes. The interior of this structure undergoes gradual acidification to activate lysosomal hydrolases, which further degrade the encapsulated contents. Dysfunctional autophagy is tightly linked to a variety of pathological processes including tumors, neurodegenerative diseases, metabolic disorders, and pathogen infections. Given the critical role of cellular autophagy in physiological and pathological processes, it has become a research hotspot in cell biology. The autolysosome serves as the final effector structure and key functional hub of the autophagic process. Therefore, detecting autolysosome formation and its functional status is an important indicator for evaluating the integrity and efficiency of autophagy, and is also of great significance for studying diseases triggered by defective autophagy.

AL-Green (Autolysosome Green) is a small-molecule lipophilic fluorescent probe with excellent cell membrane permeability. After entering cells, AL-Green is encapsulated within autophagosomes. When autophagosomes fuse with lysosomes to form autolysosomes, the luminal environment becomes acidified. AL-Green is highly sensitive to acidic conditions; its fluorescence signal increases markedly with rising acidity, thereby emitting intense fluorescence selectively within autolysosomes. Leveraging this property, AL-Green enables specific labeling of autolysosomes and real-time visualization of autolysosome formation. It is an important tool for monitoring autophagosome‑lysosome fusion and subsequent degradation. In addition, we offer the Autophagosome Fluorescence Staining Kit (AP-Green) for monitoring cellular autophagy.

Based on the staining principle of AL-Green, this kit is only intended for fluorescence staining and detection of autolysosomes in live cells. It cannot be used for staining frozen or fixed cells, tissues, or tissue sections.

Following staining with this kit, imaging can be performed using a fluorescence microscope, and fluorescence measurement can also be conducted via confocal microscope or flow cytometer. For fluorescence microscopy, UV-range excitation light is used to elicit green fluorescence. For confocal microscopy, the recommended excitation wavelength is 370 nm (range: 350–450 nm) and emission wavelength is 510 nm (range: 500–560 nm). For flow cytometry, the KO525 channel (Ex/Em = 405/525 nm) can be used for detection.

AutophagyUp (1000×) supplied in this kit can be used as a positive control to induce cellular autophagy and subsequent autolysosome formation. The working concentration is generally 0.1–5×, with 1× as the preferred recommended final concentration.

Storage Conditions

Store at -20 ºC, valid for 1 year. AL-Green (1000X) must be protected from light.

Precautions

• AL-Green is extremely light-sensitive. Minimize light exposure during handling.
• Add AL-Green prior to autophagy inducers or inhibitors to enable real-time monitoring of autophagosome formation.
• This kit is for live cell detection only. It is not suitable for fixed or frozen cell or tissue samples.
• This product is for research use only by professionals. It shall not be used for clinical diagnosis or therapy, food or pharmaceutical applications, and must not be stored in residential premises.
• For your safety and health, wear a lab coat and disposable gloves during operation.