​ Cell Tracker CM-DiI是DiI的衍生物，在水中的溶解性比DiI好。它可以自由穿过细胞膜进入细胞，转化为细胞膜不可渗透的反应产物。Cell Tracker CM-DiI染料通过几代培养保留在活细胞中。Cell Tracker CM-DiI染料至少能稳定发出72 h的荧光，具有理想的跟踪性能，且拥有性质稳定，工作浓度无毒性，良好的细胞保留性等优点，在生理pH条件下可具有明亮的荧光。此外，Cell Tracker CM-DiI 染料的激发和发射光谱与GFP（绿色荧光蛋白）光谱充分分离，可以多种颜色复用。 Cell Tracker CM-DiI包含一个温和的巯基反应氯甲基取代基，该取代基通过与含巯基的肽和蛋白质共价结合，从而可以与含醛基的固定剂交联，固定后染色信号不会丢失。相比于DiI等非共价结合的染料，更适合于固定细胞的染色。 以每次使用100 μL染色工作液，染色工作液浓度5 μM计算，20 μg配置为工作液大概可以用38次，1 mg配置为工作液大概可以用1900次。 注意事项： 1. 使用前请将产品瞬时离心至管底，再进行后续实验。 2. 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。 3. 为了您的安全和健康，请穿实验服并戴一次性手套操作。 产品参数： Ex/Em ：553/570 nm 应用范围： 细胞膜荧光染料、细胞追踪与长期示踪 使用方法： 1. 染色液制备 （1） 配置 DMSO 或 DMF 储存液：储存液用 DMSO 或 DMF 配置成浓度 1 mM 的储液。 注：未使用的储存液分装储存在-20℃，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 1~5 μM 的工作液。 注：工作液最终浓度建议根据不同细胞系和实验体系来优化。建议从推荐浓度的 10 倍范围内开始最优浓度的摸索。 2. 细胞染色 将细胞用工作液在 37℃下孵育 60 min，标记完成后，用 PBS 清洗细胞，并在新鲜培养基中重悬。对于贴壁细胞，贴壁培养时进行标记比消化成悬液后标记，细胞生存力更高。 以下为可选步骤： Cell Tracker CM-DiI 染色兼容后续的固定和透化。 （1）固定。用 3.7%多聚甲醛（溶于 PBS），室温固定 10 min。室温条件 PBS 清洗 2 次，每次 5 min。 （2）透化。用丙酮在-20℃下透化 10 min。室温条件 PBS 清洗 2 次， 每次5 min。 ​ Cell tracker cm DiI is a derivative of DiI and has better solubility in water than DiI. It can freely cross the cell membrane and enter the cell, and transform into the reaction product that is impermeable to the cell membrane. Cell tracker cm DiI dye was retained in living cells by several generations of culture. Cell tracker cm DiI dye can stably emit fluorescence for at least 72 h, has ideal tracking performance, and has the advantages of stable property, non-toxic working concentration, good cell retention, and can have bright fluorescence under physiological pH conditions. In addition, the excitation and emission spectra of cell tracker cm DiI dye are fully separated from the GFP (green fluorescent protein) spectrum and can be reused in multiple colors. Cell tracker cm DiI contains a mild thiol reactive chloromethyl substituent, which can be cross-linked with aldehyde containing fixatives by covalently binding with thiol containing peptides and proteins, and the staining signal will not be lost after fixation. Compared with non covalently bound dyes such as DiI, it is more suitable for the staining of fixed cells. At 100 per use μ L of dyeing working solution with a concentration of 5 μ M calculation, 20 μ G can be used for about 38 times as working fluid, and 1 mg can be used for about 1900 times as working fluid. Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 3. for your safety and health, please wear experimental clothes and disposable gloves. Product parameters: Ex/Em：553/570 nm Scope of application： Cell membrane fluorescent dye, cell tracking and long-term tracing Usage: 1. Preparation of staining solution (1) Configure DMSO or DMF storage solution: Use DMSO or DMF to prepare a storage solution with a concentration of 1 mM. Note: Unused storage liquids should be packaged and stored at -20 ℃ to avoid repeated freeze-thaw cycles. (2) Preparation of working solution: Dilute the storage solution with a suitable buffer (such as serum-free medium, HBSS or PBS) and prepare a concentration of 1-5 μ M's working fluid. Note: It is recommended to optimize the final concentration of the working solution based on different cell lines and experimental systems. It is recommended to start exploring the optimal concentration within a range of 10 times the recommended concentration. 2. Cell staining Incubate the cells with working solution at 37 ℃ for 60 minutes. After labeling, wash the cells with PBS and resuspend them in fresh culture medium. For adherent cells, labeling during adherent culture is more effective than labeling after digestion into suspension, resulting in higher cell viability. The following are optional steps: Cell Tracker CM DiI staining is compatible with subsequent fixation and translucency. (1) Fixed. Fix at room temperature for 10 minutes using 3.7% paraformaldehyde (soluble in PBS). Clean twice with PBS at room temperature for 5 minutes each time. (2) Translucency. Permeate with acetone at -20 ℃ for 10 minutes. Clean twice with PBS at room temperature, each time 5 min.