Fluorescein di-β-D-glucuronide is a fluorogenic substrate with an excitation wavelength of 488nm and an emission wavelength of 530nM, which is widely used to detect β-galactosidase activity in single cells and microcolonies via flow cytometry[1][2]. Fluorescein di-β-D-glucuronide is hydrolyzed by β-galactosidase in a two step process, first to fluorescein-mono-β-D-galactopyranoside (FMG), which is slightly fluorescent, and then to highly fluorescent fluorescein[3].
In vitro, Fluorescein di-β-D-glucuronide(182µM) is used to incubate to formaldehyde-fixed Hs68 human foreskin fibroblasts for 24h at 37°C in the dark without CO₂. Fluorescein di-β-D-glucuronide was able to enter cells, causing fluorescence emission proportional to the amount of β-galactosidase[4].
References:
[1] Nir R, Yisraeli Y, Lamed R, Sahar E. Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity. Appl Environ Microbiol. 1990 Dec;56(12):3861-6.
[2] Plovins A, Alvarez A M, Ibañez M, Molina M, Nombela C. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells.Appl Environ Microbiol. 1994 Dec;60(12):4638-41.
[3] Hofmann J, Sernetz M. A kinetic study on the enzymatic hydrolysis of fluorescein diacetate and fluorescein-di-beta-D-galactopyranoside. Anal Biochem. 1983 May;131(1):180-6.
[4] Yang N C, Hu M L. 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.
Fluorescein di-β-D-glucuronide是一种荧光底物，激发波长488nm，发射波长530nm，广泛用于流式细胞术检测单细胞和微菌落中的β-半乳糖苷酶活性[1][2]。Fluorescein di-β-D-glucuronide被β-半乳糖苷酶两步水解：先生成弱荧光的fluorescein-mono-β-D-galactopyranoside（FMG），再生成高荧光强度的荧光素[3]。
体外实验中，将182µM Fluorescein di-β-D-glucuronide与经甲醛固定的Hs68人包皮成纤维细胞在37°C、避光、无CO₂条件下孵育24h。Fluorescein di-β-D-glucuronide可进入细胞，其荧光强度与β-半乳糖苷酶含量成正比[4]。