A fluorimetric method using fluorescein di-β-D-galactopyranoside for quantifying the senescence-associated β-galactosidase activity in human foreskin fibroblast Hs68 cells

The 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.