PFB-FDGlu (5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside)

PFB-FDGlu is cell-permeable and when loaded into cells, localizing to endosomal and lysosomal compartments while maintaining cell viability. Upon enzymatic cleavage by active GCase enzymes, it yields a green-fluorescent dye (PFB-F) with excitation/emission maxima of approximately 492/516 nm. This fluorescence can be detected using fluorescence microscopy, high-content imaging, and flow cytometry, allowing for specific, single-cell analysis of GCase activity.

Advantages of PFB-FDGlu for detecting GCase activity in lysosomes:

• High specificity—utilizes a lysosomal GCase substrate for measurement of GCase activity, ensuring specific lysosomal staining (note that with long incubations the fluorescent PFB-F will migrate to the cytoplasm)
• Live cell imaging—allows for the assessment of GCase activity in intact, living cells with minimal impact on cell viability, even with extended incubation
• Single-cell analysis—enables specific, single-cell analysis with flow cytometry, high-content imaging, and microscopy to distinguish between different cell types
• Bright fluorescence—yields a bright green fluorescent dye upon cleavage by GCase, enhancing detection sensitivity
• Versatile protocol—can be combined with other phenotypic indicators, such as viability stains and antibodies, to identify distinct cell populations or as a selection criterion for cell sorting

GCase, encoded by the GBA1 gene, is a lysosomal hydrolase that converts glucosylceramide to glucose and ceramide. Mutations in GBA1 leading to GCase deficiency can cause lysosomal storage disorders. GCase deficiencies are implicated in several diseases, including Gaucher's disease and Parkinson's disease.