Lysosomal Activity Assay Kit
Although the intracellular activity of lysosome is difficult to measure in living cells, Assay Genie has developed a proprietary Lysosome-Specific Self-Quenched Substrate which has low background fluorescence, high signal to background ratio and is pH insensitive. The substrate, acting as endocytic cargo, can be taken up by cells and degraded within an endo-lysosomal vesicle. The fluorescent signal is recovered from the Self-Quenched Substrate. The fluorescence signal, generated by degradation, is proportional to the intracellular lysosomal activity and can be measured using a fluorescence microscopy and/or flow cytometry. The cell based Lysosomal Intracellular Activity Assay Kit includes Cytochalasin D, a cell-permeable inhibitor of endocytosis that serves as an experimental control. This easy-to-use, non-radioactive kit allows imaging and accurate measurement of de-quenching substrate in cultured cells.
|Product Name:||Lysosomal Activity Assay|
|Detection Method:||Flow Cytometry (FL1)/ Microscopy (Ex: 488 nm)|
|Sample Type:||Suspension or adherent cells cultures|
|Storage:||Store the kit at -20°C, protect from light|
|Shipping Conditions:||Gel pack|
|Usage:||For research use only. Not for use in humans.|
Lysosomal Activity Assay Kit Components
|Assay Buffer (50X)||1.8 ml|
|Self-Quenched Substrate||1 vial|
|Cytochalasin D (100X)||50 µl|
Figure: Release of self-Quenched Substrate in U937 cells. 1x10 6 U937 cells were pretreated with vehicle or 1X Cytochalasin D for 1 hr. After pre-treatment, cells were incubated with Self-Quenched Substrate and the same concentration of Cytochalasin D for additional hour in medium supplemented with 0.5% FBS according to kit’s protocol. Panel A: Comparison of histograms from flow analysis showing the inhibition of De-Quenching of Substrate by Cytochalasin D. Unstained cells (black); experimental control (green) in the presence of 1X Cytochalasin D; Positive control (pink) without 1X Cytochalasin D. Panel B: Images of U937 cells obtained using fluorescence microscope. Top: positive control cells treated Self-quenched substrate only. Bottom: negative control cells treated with 1X Cytochalasin D. U937 cells showing the release of Self-quenched substrate in the endocytotic vesicle (punctured pattern). Panel C: Lysosomal staining with Lysosomal Associated Membrane Protein 1 (Lamp-1 RFP, a lysosomal marker). Cells were stained with nuclear dye for 10 min, washed with 1X PBS and mounted on the slide. Images were taken using a fluorescence microscope with a 60X objective lens.
Lysosomes are membrane-bound organelles important for various cellular processes. They contain hydrolytic enzymes that are utilized in the metabolism of some biomolecules. The extracellular cargo (e.g. nutrients toxins) binds to the cell membrane and is subsequently transported into membrane-bound endosomes for further degradation by lysosomes while intracellular components are transported to lysosomes through autophagy. Lysosomal dysfunction is associated with many human conditions such as aging and neurodegenerative disease.