FAβ-gal: an automated fluorescence-based quantification of the senescence-associated beta-galactosidase X-gal assay

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.