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by claude@2026-07, 2026-07-03
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The paper introduces FAβ-gal, an automated, fluorescence-based quantification method for senescence-associated β-galactosidase activity that uses far-red fluorescence from the X-gal cleavage product indigo as a measurable readout on conventional wide-field fluorescence microscopy. Using workflow and software tools, it standardizes SA-β-gal quantification in a semiautomated, unbiased way and reports that FAβ-gal measurements show a strong linear correlation with the percentage of senescent cells and high sensitivity, with applicability to tissue sections as well. A stated limitation is that the method is designed to address the challenging quantification of the original colorimetric SA-β-gal assay rather than to replace its underlying biology, and the demonstrated use includes DNA damage–induced senescence in U2OS tumor cells. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
Cellular senescence plays a pivotal role in aging and cancer, two major biomedical and socioeconomic challenges of our time. Therefore, its study has become crucial for the design of interventions based on its manipulation. In this sense, researchers have developed a wide variety of techniques to detect and quantify cellular senescence. Among them, the most popular is the original Senescence-Associated β-galactosidase (SA-β-gal) colorimetric assay, based on the use of the chromogenic substrate X-gal. This compound is cleaved by β-galactosidase, producing an insoluble, blue precipitate of 5,5’-dibromo-4,4’-dichloro-indigo (commonly referred to as indigo). While this method remains the gold standard senescence assay, its quantification remains challenging due to the color-based readout. In this work, we describe a method, which we have named FAβ-gal (Fluorescence Analysis of β-galactosidase), that exploits the far-red fluorescence of the β-gal product indigo and allows the quantification of SA-β-gal activity under any conventional wide-field fluorescence microscopy using the original X-gal assay. In addition, we developed workflows and software applications that standardize SA-β-gal quantification in a semiautomatic and unbiased manner. We demonstrate that FAβ-gal measurements present a strong linear correlation with the percentage of senescent cells and show high sensitivity. Moreover, we show that this method is also applicable to tissue sections, underscoring the versatility of our approach. Therefore, FAβ-gal could be easily introduced into the routine of laboratories already using the original colorimetric assay, enhancing the accuracy, sensitivity and reproducibility of senescence detection.
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Abstract
Cellular senescence plays a pivotal role in aging and cancer, two major biomedical and socioeconomic challenges of our time. Therefore, its study has become crucial for the design of interventions based on its manipulation. In this sense, researchers have developed a wide variety of techniques to detect and quantify cellular senescence. Among them, the most popular is the original Senescence-Associated β-galactosidase (SA-β-gal) colorimetric assay, based on the use of the chromogenic substrate X-gal. This compound is cleaved by β-galactosidase, producing an insoluble, blue precipitate of 5,5’-dibromo-4,4’-dichloro-indigo (commonly referred to as indigo). While this method remains the gold standard senescence assay, its quantification remains challenging due to the color-based readout. In this work, we describe a method, which we have named FAβ-gal (Fluorescence Analysis of β-galactosidase), that exploits the far-red fluorescence of the β-gal product indigo and allows the quantification of SA-β-gal activity under any conventional wide-field fluorescence microscopy using the original X-gal assay. In addition, we developed workflows and software applications that standardize SA-β-gal quantification in a semiautomatic and unbiased manner. We demonstrate that FAβ-gal measurements present a strong linear correlation with the percentage of senescent cells and show high sensitivity. Moreover, we show that this method is also applicable to tissue sections, underscoring the versatility of our approach. Therefore, FAβ-gal could be easily introduced into the routine of laboratories already using the original colorimetric assay, enhancing the accuracy, sensitivity and reproducibility of senescence detection.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This new version includes the results of applying our method to senescence induced by DNA damage in tumor cells (U2OS), corrects some minor errata and adds Gabriel Bretones as a coauthor.
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