Fluorescein di(β-D-galactopyranoside)

Fluorescein di(β-D-galactopyranoside) is a fluorogenic substrate for β-galactosidase (λ_ex=485 nm, λ_em=535 nm). In Vitro:Assay of senescence-associated β-galactosidase (SA-βG) activity^[1].
The cells (5×10³ cells per well) are cultured in a 96- well plate.
Wash with PBS.
Fixed for 5 min in 2% formaldehyde and 0.2% glutaraldehyde buffered with PBS.
Washing thrice in PBS.
An aliquot (100 μL) of the reaction buffer (i.e., the staining solution without X-Gal) is added into each well (containing 3.7 mL 0.2 M citric acid, 6.3 mL 0.4 M Na2HPO4 (citrate-phosphate buffer), 1 mL 100 mM potassium ferricyanide, 1 mL 100 mM potassium ferrocyanide, 0.6 mL 5 M NaCl, 0.2 mL 0.2 M MgCl2, and 7.2 mL deinozied water in a total volume of 20 mL is used) .
10 μL of 2 mM FDG is added per well and the plate was incubated in the dark at 37°C for 24 h without CO2 supply.
The fluorescein fluorescence was measured using a fluorometer (Ex/Em = 485/535 nm).