Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level.
Cells
; 13(8)2024 Apr 19.
Article
in En
| MEDLINE
| ID: mdl-38667321
ABSTRACT
BACKGROUND:
Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells.METHODS:
Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation.RESULTS:
Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation.CONCLUSION:
A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Alpha-Galactosidase
/
Flow Cytometry
Limits:
Humans
Language:
En
Journal:
Cells
Year:
2024
Document type:
Article
Affiliation country:
Hungary