​ DiD染料是亲脂性荧光染料家族成员之一，它可以用来染细胞膜和其它脂溶性生物结构。当DiD与细胞膜结合后其荧光强度大大增强，这类染料有着很高的淬灭常数和激发态寿命。一旦对细胞染色，这类染料在整个细胞膜上扩散，最佳浓度时可以使整个细胞膜染色。DiD（远红色荧光）可以用来对活细胞进行成像和流式分析。DiD可以用633 nm He–Ne激光器激发，有着比DiI（一种常见的细胞荧光染料）更长的激发波长和发射波长，在细胞和组织染色中更有价值。 DiD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。 以每次使用100 μL染色工作液，染色工作液浓度5 μM计算，10 mg配置为工作液大概可以用19009次。 产品参数： Ex/Em (MeOH) = 644/663 nm 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. DiD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。 3. 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。 4. 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；神经元顺行和逆行示踪；细胞长期示踪 使用方法： 1.染色液制备 （1）配制储液：储液用无水DMSO或EtOH配制，浓度1~5mM。 注：未使用的储存液分装储存在-20°C，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）稀释储液，配制浓度为1~5μM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.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°C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°C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°C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的效果。 （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。 ​ Did dye is a member of lipophilic fluorescent dye family, which can be used to dye cell membranes and other lipid soluble biological structures. When did is combined with the cell membrane, its fluorescence intensity is greatly enhanced. This kind of dye has a high quenching constant and excited state lifetime. Once the cell is stained, this kind of dye spreads on the whole cell membrane, and the whole cell membrane can be stained at the optimal concentration. Did (far red fluorescence) can be used for imaging and flow analysis of living cells. Did can be excited by 633 nm he – Ne laser. It has a longer excitation wavelength and emission wavelength than DiI (a common cellular fluorescent dye), which is more valuable in cell and tissstaining. The fixation of paraformaldehyde (methanol and other reagents are not allowed) can be carried out after did staining, but permeabilization 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. At 100 per use μ L of dyeing working solution with a concentration of 5 μ According to m calculation, 10 mg of working fluid can be used for 19009 times. Product parameters： Ex/Em (MeOH) = 644/663 nm Matters needing attention： 1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when did 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. 3. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves. Scope of application： Cell membrane fluorescent dye ; anterograde and retrograde tracing of neurons ; long-term cell tracing Usage: 1. Preparation of staining solution (1) Preparation of storage solution: The storage solution is prepared with anhydrous DMSO or EtOH, with a concentration of 1-5mM. Note: Unused storage liquids should be packaged and stored at -20 ° C 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. 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 ° C 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 ° C 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 ° C 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.