A lipophilic fluorescent dye
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3,3′-Dioctadecyloxacarbocyanine perchlorate is a green fluorescent lipophilic tracer, which is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes.
A fluorescent dialkyl carbocyanine dye (DiOC18(3)) is used to label muscle membranes, and the membranes accessible to the dye were observed by confocal laser scanning microscopy. A loss of cell membrane integrity results from contractile activity, enabling DiOC18(3) staining of internal membranes. The resulting staining pattern is striking and fibers with damaged cell membranes are easily distinguished from uninjured ones[1]. 3,3′-Dioctadecyloxacarbocyanine perchlorate is a cationic oxacarbocyanine dye. The absorption and fluorescence spectra of 3,3'-dioctadecyloxacarbocyanine, a cationic oxacarbocyanine dye have been studied in aqueous and nonaqueous media containing egg phosphatidylcholine (PC) as well as in different solvents of diverse nature. The results show the evidence of complex formation of the dye in the ground and in the excited states with PC. The excited state interaction of the dye with PC suggests the electron transfer from PC to dye and this is supported by photovoltage generation in a photoelectrochemical cell consisting of dye and PC in aqueous medium[2].
Reference:
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[1]. Warren GL, et al. Redistribution of cell membrane probes following contraction-induced injury of mouse soleus muscle.
[2]. Bhowmik BB, et al. Photophysical studies of 3,3' dioctadecyloxacarbocyanine dye in model biological membranes and different solvents. Chem Phys Lipids. 2001 Feb;109(2):175-83.
本方案仅提供一个指导,应根据您的具体需要进行修改。
1. DiO细胞膜染色液制备
?(1).配置DMSO或EtOH 储存液:储存液用DMSO或EtOH配置,浓度1~5mM。
注意:未使用的储存液分装保存在-20℃避光保存,避免反复冻融。
(2).工作液制备:用合适的缓冲液(如:无血清培养基,HBSS或PBS)稀释储存液,配制浓度为0.5~5μM的工作液。
注意:工作液的最终浓度是根据不同细胞和实验的经验来配制。可以从推荐浓度的十倍以上寻找最佳条件。
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2.悬浮细胞染色
(1).悬浮细胞经4°C、1000g离心3-5分钟,弃去上清液。用PBS清洗两次,每次5分钟。
(2).加入1mL的DiO工作溶液,室温孵育5-30分钟,不同的细胞最佳培养时间不同。
(3).染色细胞试管在1000~1500转离心5分钟。
(4).4°C、400g离心3-4分钟,弃去上清液。倾倒上清液,加入PBS清洗两次,每次5分钟。
(5).用无血清细胞培养基或PBS重悬细胞。通过荧光显微镜或流式细胞术观察。
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3.粘壁细胞的染色
(1).在无菌盖玻片上培养贴壁细胞。
(2).从培养基中移走盖玻片,吸出过量的培养基,将盖玻片放在潮湿的环境中。
(3).从盖玻片的一角加入100uL的染料工作液,轻轻晃动使染料均匀覆盖所有细胞。
(4).室温条件下孵育5-30分钟,不同的细胞最佳培养时间不同。
(5).吸弃染料工作液,用培养液洗盖玻片2~3次。
4.显微镜检测:DiO的激发/发射光分别为484/501nm。
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注意事项:
1)荧光染料均存在淬灭问题,请尽量注意避光,以减缓荧光淬灭。
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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