{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T04:37:25Z","timestamp":1769575045495,"version":"3.49.0"},"reference-count":32,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2024,4,19]],"date-time":"2024-04-19T00:00:00Z","timestamp":1713484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hungarian National Research, Development and Innovation Office","award":["OTKA PD_21 138521"],"award-info":[{"award-number":["OTKA PD_21 138521"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/cells13080706","type":"journal-article","created":{"date-parts":[[2024,4,19]],"date-time":"2024-04-19T04:10:15Z","timestamp":1713499815000},"page":"706","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level"],"prefix":"10.3390","volume":"13","author":[{"given":"N\u00f3ra","family":"Fekete","sequence":"first","affiliation":[{"name":"Department of Genetics, Cell and Immunobiology, Semmelweis University, 1085 Budapest, Hungary"},{"name":"For Human Genome Foundation, 1094 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luca Kamilla","family":"Li","sequence":"additional","affiliation":[{"name":"Pediatrics Centre, T\u0171zolt\u00f3 Street Department, Semmelweis University, 1085 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gergely Tibor","family":"Kozma","sequence":"additional","affiliation":[{"name":"Nanomedicine Research and Education Center, Department of Translational Medicine, Semmelweis University, 1085 Budapest, Hungary"},{"name":"SeroScience LCC, 1089 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gy\u00f6rgy","family":"Fekete","sequence":"additional","affiliation":[{"name":"Pediatrics Centre, T\u0171zolt\u00f3 Street Department, Semmelweis University, 1085 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5789-0951","authenticated-orcid":false,"given":"\u00c9va","family":"P\u00e1llinger","sequence":"additional","affiliation":[{"name":"Department of Genetics, Cell and Immunobiology, Semmelweis University, 1085 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7742-160X","authenticated-orcid":false,"given":"\u00c1rp\u00e1d Ferenc","family":"Kov\u00e1cs","sequence":"additional","affiliation":[{"name":"For Human Genome Foundation, 1094 Budapest, Hungary"},{"name":"Pediatrics Centre, T\u0171zolt\u00f3 Street Department, Semmelweis University, 1085 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1111\/imj.15100","article-title":"Treatable Lysosomal Storage Diseases in the Advent of Disease-Specific Therapy","volume":"50","author":"Peters","year":"2020","journal-title":"Intern. 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Bioengineering, 9.","DOI":"10.3390\/bioengineering9120745"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/8\/706\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:30:44Z","timestamp":1760106644000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/8\/706"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,19]]},"references-count":32,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["cells13080706"],"URL":"https:\/\/doi.org\/10.3390\/cells13080706","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,19]]}}}