C12FDG is the fluorescent substrate of β-galactosidase. By enzymolysis of β-galactosidase, fluorescein is released and its fluorescence can be used to quantify the activity of β-galactosidase. The excitation/emission maxima of fluorescein are 485/530 nm[1-3].
C12FDG(15 μM ; 2 h) staining can assess SA-β-Gal activity to detect senescence in THP-1 cells(15 μM ; 2 h) [4]and A172 cells(90 min; 33 µM) [5]. Live cell measurements of senescence-associated β-galactosidase (SA-β-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs [6].
References:
[1]. Zhang YZ, Naleway JJ, et,al. Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates. FASEB J. 1991 Dec;5(15):3108-13. doi: 10.1096/fasebj.5.15.1720751. PMID: 1720751.
[2]. Plovins A, Alvarez AM, et,al. 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. doi: 10.1128/aem.60.12.4638-4641.1994. PMID: 7811104; PMCID: PMC202038.
[3]. Fuhrmann-Stroissnigg H, Santiago FE, et,al. SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs. J Vis Exp. 2019 Jun 28;(148). doi: 10.3791/58133. PMID: 31305507.
[4]. Díaz M, Pibuel M, et,al. 4-Methylumbelliferone induces antitumor effects independently of hyaluronan synthesis inhibition in human acute leukemia cell lines. Life Sci. 2021 Dec 15;287:120065. doi: 10.1016/j.lfs.2021.120065. Epub 2021 Oct 19. PMID: 34678263.
[5]. Beltzig L, Frumkina A, et,al. Cytotoxic, Genotoxic and Senolytic Potential of Native and Micellar Curcumin. Nutrients. 2021 Jul 13;13(7):2385. doi: 10.3390/nu13072385. PMID: 34371895; PMCID: PMC8308652.
[6].Waltz RA, Whitney KE, et,al. A Systemic and Local Comparison of Senescence in an Acute Anterior Cruciate Ligament Injury-A Pilot Case Series. Life (Basel). 2023 Jul 15;13(7):1567. doi: 10.3390/life13071567. PMID: 37511942; PMCID: PMC10381817.
C12FDG是β-半乳糖苷酶的荧光底物。通过酶解β-半乳糖苷酶释放荧光素，荧光素的荧光可以用来定量测定β-半乳糖苷酶的活性。荧光素的激发/发射最大值为485/530 nm[1-3]。
C12FDG (15 μm;2 h)染色可评估SA-β-Gal活性，检测THP-1细胞(15 μM;2 h) [4]和A172细胞(90 min;33μM) [5]。使用荧光底物C12FDG测量衰老相关β-半乳糖苷酶(SA-β-Gal)活性，结合使用DNA插入Hoechst染料测定细胞总数，为筛选老年治疗药物提供了可能[6]。