Abstract
Senescence is an important cellular program occurring in development, tissue repair, cancer, and aging. Increased senescence is also associated with disease states, including obesity and Type 2 diabetes (T2D). Characterizing and quantifying senescent cells at a single cell level has been challenging and particularly difficult in large primary cells, such as human adipocytes. In this study, we present a novel approach that utilizes reflected light for accurate senescence-associated beta-galactosidase (SABG) staining measurements, which can be integrated with immunofluorescence and is compatible with primary mature adipocytes from both human and mouse, as well as with differentiated 3T3-L1 cells. This technique provides a more comprehensive classification of a cell's senescent state by incorporating multiple criteria, including robust sample-specific pH controls. By leveraging the precision of confocal microscopy to detect X-gal crystals using reflected light, we achieved superior sensitivity over traditional brightfield techniques. This strategy allows for the capture of all X-gal precipitates in SABG-stained samples, revealing diverse X-gal staining patterns and improved detection sensitivity. Additionally, we demonstrate that reflected light outperforms western blot analysis for the detection and quantification of senescence in mature human adipocytes, as it offers a more accurate representation of SABG activity. This detection strategy enables a more thorough investigation of senescent cell characteristics and specifically a deeper look at the relationship between adipocyte senescence and obesity associated disorders, such as T2D.