DiI is a long-chain carbocyanine dye. Carbocyanine dyes are widely used as Di to label cells, organelles, liposomes, viruses and lipoproteins^[2].

碳菁染料广泛用作亲脂性示踪剂，用于标记细胞、细胞器、脂质体、病毒和脂蛋白。长链碳菁包括 DiO (DiOC18(3))、DiI (DiIC18(3))、DiD (DiIC18(5)) 和 DiR，以及二烷基氨基苯乙烯基染料 DiA (4-Di-16-ASP) 用于标记膜和其他疏水结构。DiIC16(3) 的烷基取代基 (C16) 比 DiI (C18) 短。它们在脂类环境中具有极高的消光系数、依赖于环境的荧光和较短的激发态寿命。它们在水中呈弱荧光，但在与膜结合或与亲脂性生物分子结合时具有高荧光性和相当的光稳定性。这些光学特性使其成为细胞质膜染色的理想选择。一旦应用于细胞，这些染料在质膜内横向扩散，导致整个细胞染色。
DiO、DiI、DiD 和 DiR 分别显示出明显的绿色、橙色、红色和红外荧光，从而促进活细胞的多色成像和流式细胞术分析。DiO 和 DiI 可分别用于标准 FITC 和 TRITC 的荧光通道。其中，DiI 及其衍生物最常用，因为它们通常表现出非常低的细胞毒性。此外，DiI 广泛用于测定 LDL 和 HDL 等脂蛋白。亲油性氨基苯乙烯染料 DiA 也常用于神经元示踪。
制备 Di 染色溶液
1.1 制备 DMF、DMSO 或乙醇储备溶液：储备溶液应在 1-5 mM 的二甲基甲酰胺 (DMF) 、二甲基亚砜 (DMSO 或乙醇 DMSO 中制备。DMF 比乙醇更适合作为 Di 的溶剂。储备溶液应及时使用。任何未使用的溶液可作分装储存在 -20℃。避免反复冻融，溶液可储存 6 个月。
1.2 制备工作溶液：将储备溶液稀释到合适的缓冲液中，如无血清培养基、HBS 或 PBS，以制备 1-5 μM工作溶液，现配现用。
注：应根据经验确定不同实验条件下工作溶液的最终浓度。
2. 悬浮细胞
2.1 悬浮细胞：离心收集细胞，加入 PBS 洗涤两次，每次 5 分钟。细胞密度在 1×10⁶/mL
2.2 加入 1 mL Di 工作液，室温孵育 5-30 分钟。
2.3 400 g，4℃ 离心 3-4 分钟，弃去上清。
2.4 加入 PBS 洗涤细胞两次，每次 5 分钟。
2.5 用 1 mL 无血清培养基或 PBS 重悬细胞后，使用荧光显微镜或流式细胞仪进行观察。
3. 贴壁细胞
3.1 将贴壁细胞培养于无菌盖玻片上。
3.2 从培养基中移出盖玻片，吸除多余培养基。
3.3 加入 100 μL 染料工作液，轻轻晃动使其完全覆盖细胞，孵育 5-30 分钟。
3.4 吸去染料工作液，用培养基洗 2-3 次，每次 5 分钟，使用荧光显微镜或流式细胞仪进行观察。

DiI-labeled motoneurons have remained viable for up to 4 weeks in culture and up to one yearin vivo^[1]

933.87

Solid

C₅₉H₉₇ClN₂O₄

41085-99-8

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)

DMSO : 12.5 mg/mL(13.39 mM;ultrasonic and warming and heat to 80℃)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80℃, 6 months; -20℃, 1 month (sealed storage, away from moisture and light)。-80℃ 储存时，请在 6 个月内使用，-20℃ 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照In Vitro方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40%PEG300   5%Tween-80   45% saline

  Solubility: 1.25 mg/mL (1.34 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.25 mg/mL (1.34 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 12.5 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH2O 中，得到澄清透明的生理盐水溶液
• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20%SBE-β-CDin saline)

  Solubility: 1.25 mg/mL (1.34 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.25 mg/mL (1.34 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 12.5 mg/mL 的澄清 DMSO 储备液加到 900 μL20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• 
  Description: DiI is a lipophilic carbocyanine fluorescent dye with red fluorescence, it can be used to label cell membrane and liposome.
  Method: For cell membrane labeling.
  1. Cells are first incubated at 37℃ in a 5% CO₂atmosphere for 10 min and then washed three times with PBS.
  2. Fix cells in 4% paraformaldehyde at room temperature for 30 min, the cells are stained with testing antibodies in 1:250 dilutions at 4℃ overnight and then wash cells three times with cold PBS.
  3. Following washing, the cells are stained with a secondary antibody at 1:300 dilutions for 60 min at room temperature.
  4. Incubate cells with DiI for 15 min.
  5. Use a confocal laser microscope for image.
• 
  Description: DiI is a lipophilic carbocyanine fluorescent dye with red fluorescence, it can be used to label cell membrane and nuclei.
  Method: For cell membrane and nuclei labeling.
  1. Cultured cells are washed three times with PBS and fixed in 4% paraformaldehyde at room temperature for 30 min.
  2. Fixed cells are labeled with an antibody (1:500) at 4℃ overnight and then washed for three times with cold PBS.
  3. Cells are stained with a secondary antibody (1:500) for 1 hour at room temperature. At the same time, the cell membrane and nuclei are stained with DiI (10 min).
  4. Pictures are obtained with a confocal microscope (FV1000, Olympus, Japan).
• 
  Description: DiI is a lipophilic carbocyanine fluorescent dye with red fluorescence, it can be used to label cell membrane for long-term monitoring of cells.
  Method: For cell membrane labeling.
  1. Before imaging, cells are fixed with 4% paraformaldehyde (4 min).
  2. For 3D imaging, stain cells with DiI (5 μM; 6 min) after the cell culture is removed.
  3. Then fix cells are fixed and the fluorescent images are acquired with an A1⁺confocal fluorescence microscope (Nikon, Japan).
• 
  Description: DiI is a lipophilic carbocyanine fluorescent dye with red fluorescence, it can be used to label cell membrane for the immunofluorescence staining of exosomes.
  Method: For cell membrane labeling.
  1. Before imaging, cells are fixed with 4% paraformaldehyde (4 min).
  2. For 3D imaging, stain cells with DiI (5 μM; 6 min) after the cell culture is removed.
  3. Then fix cells are fixed and the fluorescent images are acquired with an A1⁺confocal fluorescence microscope (Nikon, Japan).
• 
  Description: DiI is a lipophilic carbocyanine fluorescent dye with red fluorescence, it can be used to label cell membrane for exosomes uptake assay.
  Method: For cell membrane labeling.
  1. Exosomes are labeled with DiI and the labeled exosomes are ultracentrifugation at 100,000 g for 70 min (twice).
  2. The exosomes pellets are washed with PBS followed by the second ultracentrifugation at 110,000 g for 70 min at 4℃ and then resuspended in PBS.
  3. The exosomes (10 μg/mL) are incubated with recipient cells per well in 24-well plate containing round coverslips for 4 and 8 h at 37℃ under 5% CO₂in the air, respectively.
  4. Wash cells with PBS for twice, then fixed with 4% paraformaldehyde for 10 min, and wash cells with PBS for three times.
  5. Images are captured with a stereofluorescence microscope (Nikon Eclipse Ni, Toyko, Japan).