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Fiji-Based Tool for Rapid and Unbiased Analysis of SA-β-Gal Activity in Cultured Cells.

ABSTRACT: Normal cells under stressful conditions such as DNA damage or excessive mitogenic signaling may undergo senescence, which is associated with cell cycle arrest and induction of a proinflammatory phenotype. Accumulation of senescent cells may contribute to the shortening of the life span by accelerating aging and promoting chronic diseases. Cytochemical detection of the senescence-associated β-galactosidase (SA-β-gal) activity with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) is a widely recognised marker of cell senescence. However, its simplicity and cost effectiveness lead to limitations in quantification, which is usually limited to manual counting of the positive cells. In order to address those limitations, we developed a Fiji-based macro extension that performs automatic and unbiased analysis of the integrated density of SA-β-gal specific signal. Our tool is not only faster than manual counting but also provides extra resolution compared to the manual methods. Our macro extension could be a valuable tool in any senescence research laboratory.

SUBMITTER: Krzystyniak A

PROVIDER: S-EPMC9953415 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Fiji-Based Tool for Rapid and Unbiased Analysis of SA-β-Gal Activity in Cultured Cells.

Krzystyniak Adam A Gluchowska Agata A Mosieniak Grazyna G Sikora Ewa E

Biomolecules 20230214 2

Normal cells under stressful conditions such as DNA damage or excessive mitogenic signaling may undergo senescence, which is associated with cell cycle arrest and induction of a proinflammatory phenotype. Accumulation of senescent cells may contribute to the shortening of the life span by accelerating aging and promoting chronic diseases. Cytochemical detection of the senescence-associated β-galactosidase (SA-β-gal) activity with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) is a wide ...[more]

PMID: 36830731

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Project description:A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies.

Dataset Information

Fiji-Based Tool for Rapid and Unbiased Analysis of SA-β-Gal Activity in Cultured Cells.

Publications

Fiji-Based Tool for Rapid and Unbiased Analysis of SA-β-Gal Activity in Cultured Cells.

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