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. 2020 Oct 17;1(3):100132.
doi: 10.1016/j.xpro.2020.100132. eCollection 2020 Dec 18.

Protocol for Probing Regulated Lysosomal Activity and Function in Living Cells

L V Albrecht  1 , N Tejeda-Muñoz  1 , E M De Robertis  1
Affiliations

Protocol for Probing Regulated Lysosomal Activity and Function in Living Cells

L V Albrecht et al. STAR Protoc. .

Abstract

Lysosomes are the catabolic center of the cell. Limitations of many lysosomal tracers include low specificity and lack of reliable physiological readouts for changes in growth factor-regulated lysosomal activity. The imaging-based protocols described here provide insights at the cellular level to quantify functions essential to lysosomal biology, including β-glucosidase enzymatic cleavage, active Cathepsin D, and pH regulation in real time. These optimized protocols, applied in different cell types and pathophysiologic contexts, provide useful tools to study lysosome function in cultured living cells. For complete details on the use and execution of this protocol, please refer to Albrecht et al. (2020).

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
LysoLive Traces β-Glucosidase Activity Where Enzyme Substrate Fluoresces upon Degradation in Lysosomes of Living Cells (A–C) Images above depict representative example of regulated-lysosomal activity in Hepatocellular Carcinoma HCC cells, which can be quantified using ImageJ and the protocols described here. Error bars denote SEM (n ≥ 3) (∗∗p>0.01). Scale bar is 10 μm.
Figure 2
Figure 2
SiR-Lysosome Traces Cathepsin D Activity in Living Cells and Can Be Monitored by Live-Cell Imaging (A and B′) Images here are stills of a movie taken of SiR-Lysosome in HCC cells. Left images are in DIC while right images are from the red fluorescence filter. Images were captured with an exposure time of 500 ms and a light intensity of 95%. See corresponding Methods Video S1. Scale bar, 10 μm.
Figure 3
Figure 3
LysoSensor Traces Lysosomal pH Gradients in Living Cells (A–C) Images depicted here demonstrate an increase in lysosomal activity visualized as increased yellow fluorescence following treatment with GSK3 inhibitor, CHIR, in HeLa cells. Yellow fluorescence can be quantified as a marker of lysosomal activity. Error bars denote SEM (n ≥ 3) (∗∗p > 0.01). Scale bar, 10 μm.
Figure 4
Figure 4
Representative Example of the Steps for Quantifying Fluorescence of Intracellular Organelles Relative to Total Cell Areas Acquire images in both DIC and in fluorescent channels of interest. DAPI (blue) can also be useful as a reference for counting total cell numbers per frame in alternative types of quantification. Right panels contain a pharmaceutical inhibitor and act as a negative control for the tracer of choice. Scale bar, 10 μm.
See this image and copyright information in PMC

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