CFDA (5(6)-Carboxyfluorescein diacetate) 活细胞示踪探针

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产品关键词：

5(6)-CFDA；Fluorescein diacetate (FDA) 二乙酸荧光素；；Calcein AM钙黄绿素；CDCFDA； CAS NO：124387-19-5；

产品信息

产品描述

CFDA（5(6)-Carboxyfluorescein diacetate，CAS：124387-19-5），一种细胞膜渗透性的酯酶底物，用作一种活细胞探针，不仅能用来测定激活其荧光所需的酶活性，也能用来检测维持荧光产物保留在胞内的细胞膜完整性。一旦被胞内酯酶水解后，代谢产生羧基荧光素（Carboxyfluorescein, CF）。与荧光素相比，羧基荧光素含有更多的负电荷，从而能更好的保留在胞内。

产品特性

1）   CAS NO：124387-19-5

2）   化学名：Spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-ar-carboxylic acid, 3',6'-bis(acetyloxy)-3-oxo-

3）   同义名：5(6)-CFDA; 5(6)-CFDA, mixed isomers;5-(and-6)-Carboxyfluorescein Diacetate, mixed isomers; 5(6)-Carboxy-di-O-acetylfluorescein; 5(6)-羧基荧光素二乙酸酯；

4）   分子式：C25H16O9

5）   分子量：460.39 g/mol

6）   外观：浅黄色至深黄色粉末

7）   纯度：≥90%（HPLC）

8）   EX/Em：492/517nm (0.1 M Tris pH 8.0 (esterase))

9）   溶解性：溶于DMSO

10）化学结构式：

保存与运输方法

保存：2-8℃避光干燥保存，长期不用可置于-20℃避光干燥保存，至少2年有效。

运输：室温运输。 

常见应用

CFDA（5(6)-Carboxyfluorescein diacetate，CAS：124387-19-5）常用来检测细菌活性和代谢活性。多与细胞核染料碘化丙啶（PI）或DAPI联合使用，以下列出此探针的一些应用文献，仅供交流学习用。

另外有资料显示，革兰氏阴性菌用CFDA染色所得的荧光强度会低于革兰氏阳性菌，原因在于，革兰氏阴性菌的外膜结构会在一定程度上阻挡CFDA进入细胞。对于革兰氏阴性菌染色，可使用添加含EDTA的染色缓冲液（比如0.1M-Phosphate buffer (pH8.5), 5%(w/v) NaCl, 0.5 mM EDTA）或戊二醛（参考文献：Morono Y et al. Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate. Biotechnol Lett. 2004 Mar;26(5):379-83.）

文献1）Yamaguchi N et al. Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods. Nephrol Dial Transplant. 2007 Feb;22(2):612-6. Epub 2006 Nov 22.

检测背景：CFDA是一种比较流行的、灵敏的荧光底物用于预估活微生物（细菌、酵母或真菌）的酯酶活性。细胞质内的酯酶活性是微生物生理活性的一种指标。通过CFDA-DAPI双染色方法同时确定总细菌数和具有生理活性的细菌数（具有酯酶活性）。

检测对象：500ml透析液样本的细菌细胞

检测方法：Bacterial cells in 500 ml of dialysis fluid samples were vacuum-filtered onto polycarbonate black filters and then incubated in staining buffer [0.1 mol/l phosphate buffer (pH8.5), 5% (w/v) NaCl, 0.5 mmol/l EDTA (ethylenediaminetetraacetic acid)] containing DAPI (final concentration, 1 μg/ml) and CFDA (final concentration, 150 μg/ml) in a filtration funnel for 5 min at room temperature (∼25°C). The staining buffer in the funnel was then removed by vacuum filtration. The filters were mounted on glass slides with immersion oil and fluorescing cells were counted at 1000 × magnification using an E-400 epifluorescence microscope equipped with a 100 W Hg lamp.

检测结果：

Fig 1.Bacterial cells in dialysis fluid. (A) Total bacterial cells stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed under ultraviolet excitation. (B) Esterase-active cells stained with carboxyfluorescein diacetate (CFDA) and observed under blue excitation. (C) Microcolonies stained with SYBR Gold and observed under blue excitation.

文献2）Majeed M et al. Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry. PLoS One. 2018 Feb 23;13(2):e0192836.

检测对象：B. coagulans MTCC 5856 spores

检测方法：Double staining with CFDA and PI were carried out by incubating the spore suspension (10⁷/ml) with 50μM CFDA at 37°C for 60 minutes in and 10μM PI in the last 10 min. The time for CFDA staining was standardized earlier at different times and 60 minutes was found to be optimum.

检测结果：

Fig 2. Flow cytometric analysis of B coagulans MTCC 5856 spores.10 mg of dry spores were suspended in 1ml of PBS and activated for 30 min at 75°C, followed by fluorescent staining as described in the methods section. Data represents mean± standard deviation of at least 3 independent experiments. A: Gating strategy for SYTOBC ™ staining- The microbeads and the stained spores are gated separately based on unstained control B: Gating strategy for CFDA and PI staining. The dot plot represents a double staining for both CFDA and PI. The quadrant 4(Q4) represents live cells, Q1- dead cells and Q2 damaged cells. The double positive cells were not considered for enumerating the live spores. C: Representative dot plot for the FCM analysis of Live and Dead spores D: Table showing the calculation of viable spores.

注意事项

1）    本品保存过程中远离氧化剂。

2）    为了您的安全和健康，请穿实验服并戴一次性手套操作。

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 — —Written/Edited by V. Shallan【版权归MKBio懋康所有】

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