Lysosomal activity was measured using the commercial Lysosomal Intracellular Activity Assay Kit according to the manufacturer’s instructions (Abcam, ab234622). Cells were incubated with a self-quenched substrate, washed with 1 mL of ice-cold 1X assay buffer and immediately imaged. Single plane images were taken using a Leica TCS STED CW SP8 laser scanning microscope using the 63X oil-immersion objective and 1.5X zoom. Images were analyzed with FIJI/ImageJ and mean fluorescence intensity was measured for each individual lysosome. Lysosomal fluorescence intensity values proportionally correlate with the amount of lysosomal activity [86 (link)]. Intensity, lysosomal number and occupied area values were normalized to the cell area. Data are shown normalized to the control of each experiment to reduce interexperimental variability
Protocol detail
Quantifying Lysosomal Activity in Cells
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Corresponding Organization : Ikerbasque
Other organizations : Universidad de Salamanca, Biomedical Research Networking Center on Neurodegenerative Diseases, Inserm, Université Claude Bernard Lyon 1, Hospices Civils de Lyon, Karolinska Institutet, University of Massachusetts Chan Medical School, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Centre National de la Recherche Scientifique, Université de Caen Normandie, Centro de Investigaciones Biológicas Margarita Salas, University of Fribourg, Universidad de Oviedo, Karolinska University Hospital
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Variable analysis
independent variables
- None explicitly mentioned
- Lysosomal activity
- Lysosomal fluorescence intensity
- Lysosomal number
- Occupied area
- Cell area
- Positive control: Not mentioned
- Negative control: Not mentioned
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