NAE-CHERNG YANG2026-10-162026-10-162004-02-15Pure ID: 15182113Pure UUID: 2df5223a-42f9-4f44-8443-1c9af040dc3e10.1016/j.ab.2003.11.012https://www.scopus.com/pages/publications/1642497603https://scholars.csmu.edu.tw/handle/123456789/24856The senescence-associated β-galactosidase (SA-βG) assay is one of the few accepted markers of cell aging. However, the cytochemical method using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) as substrate is limited in sensitivity and is only semiquantitative. Here, we modified the X-Gal method by replacing X-Gal with fluorescein di-β-D-galactopyranoside (FDG) as substrate for SA-βG, and the activity was measured fluorimetrically. We showed in Hs68 cells that the FDG fluorescein fluorescence increased with increasing passages of the cells in parallel with the X-Gal method. A major advantage of the FDG method is that it is a quantitative method for the SA-βG activity. For example, we showed that the FDG fluorescein in p30+1 of Hs68 cells was generally stronger than that in p26+1 cells, whereas the X-Gal method gave similar results (95 and 100%) for p26+1 and p30+1 cells. The FDG method was precise with a relative standard deviation lower than 10%. We further demonstrated that FDG and X-Gal could be added simultaneously for SA-βG assay because the FDG fluorescein diffused readily through formaldehyde-fixed cell membrane and could be detected in the suspension buffer. Thus, a double-substrate method, i.e., X-Gal for rapid qualitative assay and FDG for quantitative assay, can be conducted simultaneously to provide a simple and reliable assay of SA-βG activity as a marker of cell aging.7enjournal article