C12FDG

C12FDG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。

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货号: ajci70284
CAS: 138777-25-0
别名: N-[3',6'-二(BETA-D-吡喃半乳糖基氧基)-3-氧代螺[异苯并呋喃-1(3H),9'-[9H]氧杂蒽]-5-基]十二烷酰胺,5-Dodecanoylaminofluorescein di-β-D-Galactopyranoside
分子式: C44H55NO16
分子量: 853.9
纯度: >98%
溶解度: Soluble in DMSO
储存: Store at -20°C, protect from light
库存: 现货

Background

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].

参考文献:

[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]。

Protocol

荧光老化相关β-半乳糖苷酶(SA-β-Gal)实验^[1]:

1. 培养细胞，密度为10⁵个/mL .

2. 用200 μL PBS洗涤细胞1次，然后用100 μL固定液(2%甲醛/0.2%戊二醛蒸馏水)在室温下固定5分钟。

3. 用200 μL PBS洗涤细胞2次，用100 μL 33 μM C12FDG (PBS,pH=6.0)染色10 min，再用200 μL Hoechst溶液(1 μg/mL Hoechst 33342)在PBS(pH 6.0)上染色10 min。

4. 用20倍物镜和360 nm (Hoechst 33342)和480 nm (C12FDG)的激发滤光片对细胞成像，用460 nm和535 nm的发射滤光片对细胞进行监测。

仅供参考，应根据您的实验具体需要进行修改。

用C12FDG测定衰老相关β-半乳糖苷酶活性^[2]:

采用荧光细胞透性底物C12FDG，流式细胞术检测内皮细胞衰老相关β-半乳糖苷酶活性(SA-β-gal)。

内皮细胞(ECs)用氯喹(300 μM)碱化(pH 6) 1h，然后用SA-β-gal荧光底物C12FDG (33 μM)孵育1h。
然后用冰冷的PBS洗涤细胞，用胰蛋白酶收集细胞，用CellQuest软件进行新鲜分析。设置光散射参数以消除死细胞和亚细胞碎片。
测量绿色c12荧光素信号，利用细胞群的平均荧光强度(MFI)估计SA-β-gal活性。

参考文献:

[1]. ?Udono M, Kadooka K, et,al. Quantitative analysis of cellular senescence phenotypes using an imaging cytometer. Methods. 2012 Mar;56(3):383-8. doi: 10.1016/j.ymeth.2012.02.012. Epub 2012 Mar 3. PMID: 22406489.

[2]. Qureshi AW, Altamimy R, et,al. Ageing enhances the shedding of splenocyte microvesicles with endothelial pro-senescent effect that is prevented by a short-term intake of omega-3 PUFA EPA:DHA 6:1. Biochem Pharmacol. 2020 Mar;173:113734. doi: 10.1016/j.bcp.2019.113734. Epub 2019 Dec 5. PMID: 31811867.