Peroxisome staining in mammalian cells using peroxisome-specific probes

Howman, Charlotte, Cohen, Tamar, Shlomy, Mor Yarshansky, van Aerle, Melanie S., Carmichael, Ruth E., Schuhmacher, Milena, Reymond, Luc, Zalckvar, Einat, Kaganovich, Daniel and Amen, Triana (2025) Peroxisome staining in mammalian cells using peroxisome-specific probes. Journal of Visualized Experiments, 2025 (226), [e69405]. (doi:10.3791/69405).

Abstract

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization of peroxisomes is key to identifying peroxisome dysfunction; however, progress has been limited by the lack of peroxisome-specific probes. By developing PeroxiSPY, peroxisome-specific probes that mimic natural peroxisome metabolites, we enabled real-time detection, tracking, and quantification of peroxisome dynamics in live mammalian cells. Additionally, the applications of this technique to patient cells with peroxisome disorders reveal pathological phenotypes. Here, we provide a detailed protocol for the detection of peroxisomes in live mammalian cells, as well as the detection of peroxisomes post-staining in fixed cells. We demonstrate how to stain peroxisomes in live cells using peroxisome-specific probes, focusing on the preparation of cells, timing of the procedure, reproducibility, and troubleshooting. These probes enable rapid, specific, and non-toxic live-cell detection of peroxisomes, offering a unique, quantitative method for assessing peroxisome function and its dysfunction in peroxisome-related disorders.

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Contributors

Author: Daniel Kaganovich

Author: Triana Amen

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