C12FDG (5-Dodecanoylaminofluorescein di-beta-D-Galactopyranoside) is used as a lipophilic green fluorescent substrate for detecting beta-galactose activity, a green fluorescent substrate, is a senescence marker and is often used in conjunction with flow cytometry.

Fluorescence aging-related β-galactosidase (SA-β-Gal) assay [1]:
1.Cultivate cells at a density of 10^5 cells/mL.
2.Wash cells once with 200 μL PBS, then fix with 100 μL fixation solution (2% formaldehyde/0.2% glutaraldehyde in1. distilled water) at room temperature for 5 minutes.
3.Wash cells twice with 200 μL PBS, stain with 100 μL 33 μM C12FDG (PBS, pH 6.0) for 10 minutes, then stain with 200 μL Hoechst solution (1 μg/mL Hoechst 33342) in PBS (pH 6.0) for 10 minutes.
4.Image cells using a 20x objective and excitation filters of 360 nm (Hoechst 33342) and 480 nm (C12FDG), monitor cells using emission filters of 460 nm and 535 nm.
This protocol is provided for reference and should be modified according to the specific requirements of your experiment.