5-Cyano-2,3-ditolyl Tetrazolium Chloride (CTC)

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产品关键词：

5-Cyano-2,3-ditolyl Tetrazolium Chloride (CTC) 5-氰基-2,3-二甲苯基四氮唑；INT 碘硝基氯化四氮唑；Redox dye 氧化还原染料；5-Cyano-2,3-ditolyl Tetrazolium Chloride (CTC)；死活细菌鉴定；CAS NO：90217-02-0；

产品信息

产品描述

5-Cyano-2,3-ditolyl Tetrazolium Chloride (CTC) ，中文名5-氰基-2,3-二甲苯基四氮唑，是一种灵敏的氧化还原染料（Redox dye），主要用来检测微生物的代谢活性。这一膜渗透性的染料能迅速被健康的活细胞摄取，通过呼吸活动中的电子传递被还原成一种不溶于水、发红色荧光的甲臜结晶—CTC甲臜（CTC Formazan）（见下图）。而不呼吸或呼吸能力很弱的细胞不能或仅还原更少的CTC，不产生或产生更弱的荧光产物，因此，提供一种半定量的方法来评估健康&不健康细胞。CTC本身溶于水且在水溶液中无荧光。CTC甲臜虽在低粘度的液体中无荧光，但在高粘度的液体和固体状态下发红色荧光。因此，通过荧光显微镜计数或流式细胞术分析，能够研究具呼吸活性的细胞。CTC甲臜的最大激发波长为430nm或480nm，最大发射波长为630nm。

产品特性

1）   CAS NO：90217-02-0

2）   化学名：5-cyano-2,3-bis(4-methylphenyl)-2H-tetrazolium, monochloride

3）   同义名：5-Cyano-2,3-ditolyl-2H-tetrazolium chloride; 5-Cyano-2,3-di-(p-tolyl)tetrazolium chloride; Cyanotolyl Tetrazolium Chloride; 5-氰基-2,3-二甲苯基四氮唑；5-氰基-2,3-二(4-甲基苯基)四唑氯化物；

4）   分子式：C16H14ClN5 

5）   分子量：311.77 g/mol

6）   外观：白色或浅橙色结晶性粉末

7）   溶解性：溶于水

8） 纯度：＞90%

9）   化学结构式：

保存与运输方法

保存：-20℃密封干燥保存，至少2年有效。

运输：室温运输。

应用示例（来自文献，仅做参考）

Ref：Sieracki ME, Cucci TL, Nicinski J. Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures. Appl Environ Microbiol. 1999 Jun;65(6):2409-17. doi: 10.1128/AEM.65.6.2409-2417.1999. PMID: 10347021; PMCID: PMC91356.

a）  CTC孵育混合物制备（CTC incubation mixtures）：CTC溶于去离子水制备成25mM或50mM储存液，溶液室温缓慢搅拌过夜。每次实验准备新鲜的储存液，于4℃保存，数天内使用。常规实验中CTC储存液加入培养物或天然样本内，使其最终工作浓度为5mM。孵育时间从5~40min。之后用1/10稀释的10%多聚甲醛固定终止孵育。对照样本于CTC染色前固定。这些对照样本不含明显数量的CTC阳性细胞或CTF颗粒。CTC was dissolved in deionized water to prepare 25 or 50 mM stock solution, and the solution was slowly stirred overnight at room temperature. Fresh working stock solutions were prepared for each set of experiments, stored at 4°C, and used within several days. In a typical experiment, CTC was added to a culture or natural sample at a final concentration of 5 mM. The incubation times ranged from 5 to 40 min, and incubation was ended by fixation with a 1/10 dilution of 10% paraformaldehyde (final concentration, 1%). In time course experiments subsamples were fixed at certain times. Control preparations were fixed prior to CTC incubation in each experiment; these fixed controls never contained significant numbers of CTC-positive cells or CTF particles.

b）  流式分析：488nm激发器激发，收集（波长：＞630nm）的CTF荧光信号来检测和计算CTC阳性细胞。A Becton Dickinson FACScan flow cytometer equipped with a 15-mW, 488-nm, air-cooled argon ion laser was used in this study. All sample and data analyses were done with Becton Dickinson CellQuest software. Liquid samples were either analyzed immediately or frozen in small volumes (1 ml) in liquid nitrogen after 1 h of fixation with 1% (final concentration) paraformaldehyde. Simultaneous measurements of forward light scatter (relative size), 90-degree light scatter, and CTF fluorescence emission (wavelength, >630 nm) were used to detect and enumerate CTC-positive cells. CTC-positive bacteria were detected based on red signals above a baseline threshold by separating instrument noise and true red emitted light.

c）  CTF荧光的光谱特征（Spectrum of CTF fluorescence）：CTF的最大激发光谱比较宽，从450-585nm，最大激发波长接近480nm。CTF荧光的发射峰约在610nm，与对照荧光相比波长在585-670nm之间明显增强。The excitation spectrum of CTF was relatively broad, from about 450 to 585 nm, and had a maximum near 480 nm (Fig. (Fig.1A).1A). This finding agrees with microscope observations which indicated that both blue excitation and green excitation yield CTF fluorescence. Peak CTF fluorescence emission occurred at about 610 nm, and fluorescence was significantly greater than the control fluorescence at wavelengths between about 585 and 670 nm (Fig. (Fig.1B).1B).

Fig 1. Fluorescence excitation (ex) and emission (em) spectra of CTC-positive bacteria. For the excitation spectra (A) emission was measured at 600 and 650 nm. For the emission spectrum (B) excitation was measured at 475 nm. The control spectra (spectra C) are spectra obtained for a bacterial culture to which CTC was added after the cells were killed with formalin; these spectra were the same as the spectra obtained for formalin-fixed bacteria alone.

注意事项

1）   CTC在水溶液中状态很不稳定，最好现配现用。

2）   CTC可溶于水配制成25mM母液，置于4℃避光保存，最好当天用完。CTC的常用工作浓度约2.5~5mM，孵育时间为0.5~5h，具体需要根据实际检测样本类型、微生物种类来调整浓度和孵育时间。

3）   虽大部分研究者使用磷酸缓冲液（PBS）或磷酸缓冲液来进行CTC反应，但，＞10mM以上的磷酸盐对CTC还原会产生浓度依赖性的抑制效率。

4）   当用荧光显微镜检测CTC甲臜荧光，用蓝光激发滤片和红色发射滤片来检测。当用流式细胞仪分析CTC甲臜荧光，用488nm蓝色激发起激发，结合红色区域检测即可。

5）   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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