衰老是发育、组织修复、癌症和衰老过程中发生的重要细胞程序。衰老的加剧也与疾病状态有关，包括肥胖和 2 型糖尿病 (T2D)。在单细胞水平上表征和量化衰老细胞一直具有挑战性，对于大型原代细胞（例如人类脂肪细胞）尤其困难。在这项研究中，我们提出了一种利用反射光进行准确的衰老相关β-半乳糖苷酶（SABG）染色测量的新方法，该方法可以与免疫荧光相结合，并且与来自人类和小鼠的原代成熟脂肪细胞以及分化的3T3-L1细胞。该技术通过结合多种标准（包括强大的样本特异性 pH 控制），对细胞衰老状态进行更全面的分类。通过利用共焦显微镜的精度，利用反射光检测 X-gal 晶体，我们获得了优于传统明场技术的灵敏度。该策略允许捕获 SABG 染色样品中的所有 X-gal 沉淀，揭示不同的 X-gal 染色模式并提高检测灵敏度。此外，我们证明反射光在检测和定量成熟人类脂肪细胞衰老方面优于蛋白质印迹分析，因为它可以更准确地表示 SABG 活性。这种检测策略可以更彻底地研究衰老细胞特征，特别是更深入地研究脂肪细胞衰老与肥胖相关疾病（例如 T2D）之间的关系。© 2024 作者。衰老细胞由解剖学会和约翰·威利出版

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.© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.