Ethidium Homodimer-I （溴乙啡锭二聚体I，EthD-I）

Ethidium Homodimer-I (EthD-I)是一种高亲和性的荧光核酸染料，与DNA或RNA结合后，可以使荧光增强30多倍。用于哺乳动物、细菌、酵母和真菌的染色。EthD-I带有较强正电荷，所以该染料不能穿过细胞膜进行活细胞染色，但是能穿过死细胞膜的无序区域而到达细胞核并且嵌入DNA双链从而产生红色荧光。因此EthD-I可以准确的检测溶液中的核酸或者解体细胞中的核酸，是一种较灵敏的核酸染色剂。 以贴壁细胞（盖玻片）举例，每个样本100 μL染色工作液，染色工作液浓度4 μM计算，1 mg配置为工作液大概可以用于2917个样本。 产品参数： Ex/Em (结合DNA）= 528/617 nm 使用方法： 注：该操作说明适用于大多数细胞，但不同的细胞类型、细胞密度、使用的培养基以及其他一些因素都有可能影响染色效果， 本说明仅供参考。 1. 储存液的制备：取适量DMSO加入到EthD-I中，配制成2 mM储液，该储存液可于-20℃稳定保存一年。 2. 将20 μL 2 mM储液加入到10 mL无菌的组织培养级别的D-PBS中，充分涡旋混匀，使其终浓度为4 μM（推荐浓度为0.1-10 μM ，不同细胞系建议梯度设置确定最佳染色浓度）。 3. 吸取上述配置好的工作液100-150 μL加入到细胞盖玻片上使其完全覆盖。孵育最好是在含有盖子的盘子里防止染色液挥 发。 4. 室温避光孵育30-45min，若染色液浓度过高或温度过低可适当减少孵育时间。 5. 向一个新的显微镜载玻片上加入10 μL D-PBS。 6. 使用尖镊子小心且迅速将载有细胞的盖玻片倒置加在含有D-PBS的载玻片上，为了防止染色液挥发，用干净透明的指甲油 封住载玻片四周。 7. 在荧光显微镜下观察细胞染色情况。 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。 应用范围： 细胞核染色 Ethidium homodimer-i (ethd-i) is a fluorescent nucleic acid dye with high affinity. After binding with DNA or RNA, it can enhance the fluorescence by more than 30 times. Used for staining of mammals, bacteria, yeast and fungi. Ethd-i has a strong positive charge, so the dye cannot cross the cell membrane for live cell staining, but it can cross the disordered region of the dead cell membrane to reach the nucleus and embed the DNA double strand to produce red fluorescence. Therefore, ethd-i can accurately detect nucleic acids in solution or in disintegrated cells, and is a more sensitive nucleic acid stain. Take adherent cells (coverslips) as an example, 100 for each sample μ L of dyeing working solution with a concentration of 4 μ M calculation, 1 mg of working fluid can be used for 2917 samples. Product parameters： Ex/Em (binding DNA）= 528/617 nm Usage method: Note: This operating instruction is applicable to most cells, but different cell types, cell densities, culture media used, and other factors may affect the staining effect, This instruction is for reference only. 1. Preparation of storage solution: Add an appropriate amount of DMSO to EthD-I to prepare a 2 mM storage solution, which can be stably stored at -20 ℃ for one year. 2. Add 20 μ L of 2 mM stock solution to 10 mL of sterile tissue culture grade D-PBS, vortex thoroughly, and mix until the final concentration is 4 μ M (recommended concentration is 0.1-10 μ M, gradient setting is recommended for different cell lines to determine the optimal staining concentration). 3. Take 100-150 μ L of the prepared working solution and add it to the cell cover glass to completely cover it. It is best to incubate in a dish with a lid to prevent the dye from evaporating. 4. Incubate at room temperature in the dark for 30-45 minutes. If the concentration of the staining solution is too high or the temperature is too low, the incubation time can be appropriately reduced. 5. Add 10 μ L of D-PBS onto a new microscope slide. 6. Use sharp tweezers to carefully and quickly add the cover glass containing cells upside down to the glass slide containing D-PBS. In order to prevent the dye solution from volatilizing, use clean and transparent nail oil seal around the glass slide. 7. Observe cell staining under a fluorescence microscope. 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. Scope of application： Nuclear staining

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