DiA是一种细胞膜绿色荧光染料，它在细胞膜中的扩散速度比DiO快，并且经常和DiI一起使用于细胞膜双色标记。 DiA染色后可进行多聚甲醛（不可使用甲醇等其他试剂）的固定，但不建议在染色后进行透化的过程。此外，在固定透化（室温下用0.1% TritonX-100 透化）后，也可以很好地进行质膜染色。DiA对固定细胞的染色效果比DiO好。 以每次使用100 μL染色工作液，染色工作液浓度10 μM计算，50 mg配置为工作液大概可以用63532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. DiA染色固定的细胞或组织样品时，通常使用配制在PBS中的4%多聚甲醛进行固定，使用其它不适当的固定液会导致荧光背景较高。 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。 应用范围： 细胞膜荧光染料；主要用于细胞成像，细胞示踪和追踪 参数： Ex/Em (MeOH) = 491/613 nm 使用方法: 1. 染色液制备 （1）配制储液：储液用无水 DMSO、无水 DMF 或 EtOH 配制，浓度 1~5 mM。DiA 在无水 DMSO 和无水 DMF 中的溶解度比在 EtOH 中的溶解度高。 注：a.未使用的储存液分装储存在-20℃，避免反复冻融； b.发现较难溶解时可以适当加热，并用超声处理以促进溶解。 （2）工作液制备：用合适的缓冲液（如：无血清培养基，HBSS 或 PBS）稀释储液，配制浓度为 1~30 μM 的工作液。最常用的工作液浓度为 5-10 μM。 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. 悬浮细胞染色 （1）加入适当体积的染色工作液重悬细胞，使其密度为 1×106/mL。 （2）37℃孵育细胞 2~20 min，不同的细胞最佳培养时间不同。可以 20 min 作为起始孵育时间，之后优化体系以得到均一的标记效果。 （3）孵育结束，1000~1500 rpm 离心 5 min。倾倒上清液，再次缓慢加入 37℃预热的生长培养液重悬细胞。 （4）重复步骤（3）两次以上。 3. 贴壁细胞染色 （1）将贴壁细胞培养于无菌盖玻片上。 （2）从培养基中移走盖玻片，吸走过量培养液，但要使表面保持湿润。 （3）在盖玻片的一角加入 100 μL 的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。 （4）37℃孵育细胞 2~20 min，不同的细胞最佳培养时间不同。可以 20 min 作为起始孵育时间，之后优化体系以得到均一的标记效果。 （5）吸干染料工作液，用培养液洗盖玻片 2~3 次，每次用预温的培养基覆盖所有细胞，孵育 5~10 min，然后吸干培养基。但要使表面保持湿润。 4. 结果检测 样品可在培养基中进行检测，可通过荧光显微镜成像或流式细胞仪分析。 ​ Dia is a green fluorescent dye of cell membrane. Its diffusion speed in cell membrane is faster than DIO, and it is often used with DiI for dual color labeling of cell membrane. After dia staining, paraformaldehyde (methanol and other reagents cannot be used) can be fixed, but permeabilization process after staining is not recommended. In addition, after fixed permeabilization (permeabilization with 0.1% TritonX-100 at room temperature), the plasma membrane staining can also be well performed. The staining effect of dia on fixed cells was better than that of DIO. At 100 per use μ L of dyeing working solution with a concentration of 10 μ According to the calculation of M, 50 mg of working fluid can be used 63532 times. Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when dia is used to stain fixed cells or tissue samples, 4% paraformaldehyde prepared in PBS is usually used for fixation. The use of other inappropriate fixatives will lead to high fluorescence background. 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. Scope of application： Cell membrane fluorescent dye；mainly used for cell imaging, cell tracking and tracking. Parameters: Ex/Em (MeOH) = 491/613 nm Usage: 1. Preparation of staining solution (1) Preparation of storage solution: The storage solution is prepared with anhydrous DMSO, anhydrous DMF, or EtOH at a concentration of 1-5 mM. The solubility of DiA in anhydrous DMSO and anhydrous DMF is higher than that in EtOH. Note: a. Unused storage liquids should be packaged and stored at -20 ℃ to avoid repeated freeze-thaw cycles; b. When it is difficult to dissolve, it can be heated appropriately and treated with ultrasound to promote dissolution. (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 1-30 μ M's working fluid. The most commonly used working fluid concentration is 5-10 μ M. 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. Suspension cell staining (1) Add an appropriate volume of staining solution and resuspend the cells to a density of 1 × 106/mL. (2) Incubate cells at 37 ℃ for 2-20 minutes, and the optimal culture time varies for different cells. 20 minutes can be used as the starting incubation time, and then the system can be optimized to obtain a uniform labeling effect. (3) After incubation, centrifuge at 1000-1500 rpm for 5 minutes. Pour the supernatant and slowly add the growth culture medium preheated at 37 ℃ again to resuspend the cells. (4) Repeat step (3) more than twice. 3. Staining of adherent cells (1) Cultivate adherent cells on sterile cover slips. (2) Remove the cover glass from the culture medium and absorb excess culture medium, but keep the surface moist. (3) Add 100 to one corner of the cover glass μ Gently shake the dye working solution of L to evenly cover all cells with the dye. (4) Incubate cells at 37 ℃ for 2-20 minutes, and the optimal culture time varies for different cells. 20 minutes can be used as the starting incubation time, and then the system can be optimized to obtain a uniform labeling effect. (5) Dry the dye working solution, wash the cover glass with culture medium 2-3 times, cover all cells with preheated culture medium each time, incubate for 5-10 minutes, and then dry the culture medium. But keep the surface moist. 4. Result detection The sample can be detected in the culture medium and analyzed by fluorescence microscopy imaging or flow cytometry.