Cell experiment:

The cells (5×103 cells per well) are cultured in a 96-well plate overnight for attachment, washed, and then fixed in solutions. An aliquot (100 μL) of the reaction buffer (i.e., the staining solution without X-Gal) is added into each well. Then, 10 μL of 2 mM Fluorescein di(β-D-galactopyranoside) is added per well and the plate is incubated in the dark at 37℃ for 24 h without CO2 supply. After incubation at 37℃ for 24 h, 100 μL of the supernatant is transferred to a 96-well plate for fluorescent measurement in triplicates. The fluorescein fluorescence is measured using a fluorometer with an excitation at 485 nm and an emission at 535 nm[1].

Fluorescein di(β-D-galactopyranoside) is a fluorogenic substrate for β-galactosidase (λex=485 nm, λem=535 nm).

The fluorescence produced by Fluorescein di(β-D-galactopyranoside) increases in a time- and dose-dependent manner. The level of fluorescence produced by the double-substrate method is much lower than that by the Fluorescein di(β-D-galactopyranoside) method. Results show that the fluorescein produced by Fluorescein di(β-D-galactopyranoside) in Hs68 cells is proportional to the number of passages[1].

[1]. Yang NC, et al. A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence-associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells. Anal Biochem. 2004 Feb 15;325(2):337-43.