A flow cytometric assay for lysosomal glucocerebrosidase

Analytical Biochemistry
H H van EsD Valerio

Abstract

A flow cytometric assay is described for the determination of glucocerebrosidase (GC) activity using fluorescein di-beta-glucopyranoside (FDGlu). Fluorescent product is formed upon intracellular hydrolysis of FDGlu and is measured in the FL1 channel of a flow cytometer. We show that the assay is specific for lysosomal beta-glucosidase or glucocerebrosidase (1) by concentration-dependent inhibition of GC activity by conditurol-beta-epoxide (CBE), a specific irreversible inhibitor; (2) by the absence of activity in fibroblasts isolated from patients with Gaucher disease; (3) correction of the biochemical Gaucher phenotype in these cells is detectable following gene transfer and can be inhibited by CBE; (4) murine fibroblasts transfected with the human GC cDNA and expressing 1.5- to 2.5-fold higher levels of human GC in in vitro assays can be distinguished from nontransfected cells in mixing experiments; and (5) preincubation of GC expressing cells with the lysosomotropic compound chloroquine leads to a loss of the GC-mediated increase in fluorescence supporting lysosomal localization of the FDGlu hydrolyzing enzyme. This flow cytometric GC assay will be useful for monitoring GC activity at the single cell level and can be used fo...Continue Reading

Citations

Oct 29, 2011·Analytical and Bioanalytical Chemistry·Omid MotabarWei Zheng
May 27, 2011·Assay and Drug Development Technologies·Kenneth J ValenzanoElfrida R Benjamin
Mar 5, 2021·Bio-protocol·Laura P HughesNicolas Dzamko
Oct 7, 2021·Movement Disorders : Official Journal of the Movement Disorder Society·Daniel YsselsteinDimitri Krainc

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