DiR Iodide (DiIC18(7)) 細胞膜深紅色熒光探針

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溫馨提示：見細胞膜熒光探針專題，選擇您想要的最適膜標記探針。

搜索關鍵詞：

細胞膜熒光探針，DiI，DiO，DiD，DiR，DiA，CM-DiI，神經示蹤，傳統細胞膜熒光探針

訂購信息：

產品描述：

DiI, DiO, DiD和DiR作為一類長鏈的親脂性二烷基碳菁類染料（Dialkylcarbocyanines）熒光染料家族，用于標記細胞膜以及其他脂溶性生物結構。作為一類環境敏感型熒光染料，當它們與膜結合或者與親脂性生物分子（例如蛋白質，雖然在水中其熒光強度很弱）結合時，其熒光強度顯著增強。一旦進入細胞后，它們在細胞內質膜中逐步擴散，于最佳濃度條件下可將整個細胞均勻染色。這些染料具很高的淬滅系數，偏光依賴性和很短的激發壽命。

四種染料呈現不同的熒光顏色，DiI（橙色熒光）、DiO（綠色熒光）、DiD（紅色熒光）、DiR（深紅色熒光），為活細胞多色彩熒光成像分析和流式細胞術提供了一種便捷的工具。DiO和DiI分別用標準FITC和TRITC濾光器檢測，可結合使用。DiD可被633 nm氦-氖（He-Ne）激光激發，具有比DiI更長的激發和發射光波長，特別適合用于標記具本底熒光的細胞和組織。而，DiR在體內活體成像或者示蹤中意義非凡，因其所發射的紅外光可以高效地穿過細胞和組織，并且在紅外光范圍內，其本底熒光水平很低。

本品以DiR的碘鹽形式提供，英文名：1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide，純度≥95%，適用于熒光檢測研究。

基本特性：

1）同義名：DiR Iodide; DiIC18(7);1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide;

2）推薦濾光器：Omega-XF112, Chroma-41009

3）分子式：C63H101IN2

4）分子量：1013.39g/mol

5）外觀：藍色至深藍色固體

6）純度：≥95%（HPLC） 

7）Ex/Em：750/780 nm（甲醇）

8）溶解性：溶于DMF，DMSO，甲醇

9）化學結構圖：    

保存與運輸方法

保存：-20oC避光干燥保存，有效期二年。

運輸：冰袋運輸。

應用示例（來自文獻）

文獻來源：Eisenbl?tter M et al. In Vivo Optical Imaging of Cellular Inflammatory Response in Granuloma Formation Using Fluorescence-Labeled Macrophages. J Nucl Med. 2009 Oct;50(10):1676-82.

標記方法（Labeling Protocol）：脂質示蹤劑DiR（1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide），最大激發或者發射波長位于近紅外區域（Ex/Em：750/782nm），溶于乙醇（0.98mM）。預實驗中用梯度濃度的DiR（0.98~197nM）來確定標記效率。后續實驗中，單核細胞（1 × 105/mL培養基）加入2ul DiR標記液（終濃度為19.7uM）孵育5min，PBS清洗3次后，重懸于細胞培養液中。流式細胞儀檢測標記效率。

Cutaneous Granuloma (CG) Model（皮膚肉芽腫體內模型）：在小鼠側區皮下注射1ml 聚丙烯酰胺凝膠（PAG）誘導皮膚肉芽腫形成。為了誘導炎癥，向PAG內加入脂多糖LPS（10ug LPS/ml）。在凝膠移植前24h靜脈注射DiR標記的巨噬細胞或對照巨噬細胞。使用FRI和FMT在注射后每日觀察小鼠，持續7天。

FIG 1. FMT of mice showed distribution of M?s inside pellets. (A) FMT was performed 72 h after injection of labeled M?s (5 × 106) to resolve 3-dimensional distribution of M?s in target tissue (left, NIRF image; right, color-encoded FMT). FMT confirmed FRI results and showed that M?s distributed mainly in periphery of pellets. (B) Fluorescence quantification by FMT showed approximately 2.7 times higher signal in lipopolysaccharide-containing pellets (?) than in controls (□).

文獻來源：Jiang H et al. Liver-targeted liposomes for codelivery of curcumin and combretastatin A4 phosphate: preparation, characterization, and antitumor effects. Int J Nanomedicine. 2019 Mar 8;14:1789-1804.

DiR標記GA LPs的制備（Preparation of DiR-loaded GA LPs）:First, 120 mg l-α-phosphatidylcholine, 40 mg Chol, 20 mg DSPE-PEG2,000-GA, and 0.2 mg DiR were mixed in 5 mL Chol at a mass ratio of 600:200:100:1 in an eggplant-shaped flask, dried until a thin-lipid film had formed on the rotary evaporator under reduced pressure, and heated in a 35°C water-bath. The film was hydrated with 5 mL PBS, followed by sonication. The obtained DiR-GA LPs were filtered in a 220 nm filter device, and the final concentration of DiR was 40 μg/mL.

體內實時近外熒光成像（In vivo real-time near-infrared fluorescence imaging）： NIRF was used to observe the biodistribution of GA LPs formulation in vivo, and DiR-GA LPs (40 μg/mL) were prepared to monitor the fate of LPs. When tumors were 100 mm3, DiR-GA LPs were injected into tail veins of mice. Free DiR was used as control. Precisely 10 mg DiR iodide in a 10 mL volumetric bottle was dissolved in methanol at a constant volume and prepared into a 1 mg/mL drug-reserve solution, which was stored at 4°C in the refrigerator for later use. DiR iodide reserve solution diluted with normal saline to the concentration of 40 μg/mL. Mice were anesthetized with 10% chloral hydrate and real-time NIRF images obtained with excitation and emission at 745 and 835 nm, respectively.44 Results were analyzed using the Living Image 3.1 software.

FIG2. NIRF images of H22 tumor-bearing mice after injection of free DiR and DiR-GA LPs. Results showed that NIRF signals of the free-DiR group were lower than the DiR-GA LPs group from 1 to 48 hours. No fluorescence signals in the tumor regions were detected for the free-DiR group, except at 2 hours. By contrast, DiR-GA LPs increased in accumulation in tumor regions and signals were sustained at 48 hours after injection. The GA LP drug loading system increased the accumulation of DiR in tumors.

注意事項

1）熒光染料均存在淬滅問題，請盡量注意避光，以減緩熒光淬滅。

2）為了您的安全和健康，請穿實驗服并戴一次性手套操作。

使用方法

【注意】以下使用方法僅用作參考，可根據具體的實驗條件做出調整。

1. 染色液制備

1）儲存液制備：用DMSO或乙醇配置濃度1～5 mM的儲存液。例如，取25mg DiR（Mw：1013.39g/mol）溶于4.93ml無水DMSO中，充分溶解，即得到5mM的儲存液?！咀⒁狻课词褂玫膬Υ嬉悍盅b儲存在-20℃，避免反復凍融，且要避光保存。

2）工作液制備：用合適的緩沖液（如：無血清培養基，HBSS或PBS）稀釋儲存液，調整到1～5 μM的工作濃度?！咀⒁狻抗ぷ饕鹤罱K濃度需要根據不同細胞系和實驗體系來優化。建議從推薦濃度開始，以10倍范圍為區間進行最優濃度的摸索。

2. 懸浮細胞染色

1）加入適當體積的染色工作液重懸細胞，使其密度為1×106/mL。

2）37℃孵育細胞2～20min，不同的細胞最佳培養時間不同?？梢?0min作為起始孵育時間，之后優化以保證得到均一化的標記結果。

3）孵育結束，按1000～1500 rpm離心5min。

4）去除上清液，之后輕柔加入37℃預熱的生長培養液重懸細胞。

5）再重復3），4）步驟兩次。

3. 貼壁細胞染色

1）將貼壁細胞培養于無菌蓋玻片上。

2）從培養基中移走蓋玻片，濾掉過量培養液，將蓋玻片放在潮濕的小室內。

3）在蓋玻片的一角加入100μL的染色工作液，輕輕晃動使染料均勻覆蓋所有細胞。

4）37℃孵育細胞2～20min，不同的細胞最佳培養時間不同?？梢?0min作為起始孵育時間，之后優化以保證得到均一化的標記結果。

5）吸掉染色工作液，用培養液洗蓋玻片2～3次，每次用預溫的培養基覆蓋所有細胞，孵育5～10min，然后吸走培養基。

4. 顯微鏡檢測

1）DiD，DiO，DiI，DiR和DiS濾光器的選擇參見下表：

2）多色染料的同時檢測，濾光器按照以下設定：

a) DiI和DiO＝Omega XF52，Chroma 51004；

b) DiI和DiD＝Omega XF92，Chroma 51007；

c) DiI，DiO和DiD＝Omega XF93，Chroma 61005；

5. 流式細胞儀檢測

經DiO，DiI，DiD和DiR染色的細胞分別用流式細胞儀的FL1，FL2，FL3或FL4通道檢測。

引用文獻

[1]Cai J, Qian K, Zuo X, et al. PLGA nanoparticle-based docetaxel/LY294002 drug delivery system enhances antitumor activities against gastric cancer. Journal of Biomaterials Applications. 2019;33(10):1394-1406. doi:10.1177/0885328219837683

The in vivo tumor-targeting capacity of the NPs was examined using a lipophilic carbocyanine dye DiR (MaokangBio, Shanghai, China).

[2]Yuyu Zhao, et al. EpCAM Aptamer-Functionalized Cationic Liposome-Based Nanoparticles Loaded with miR-139-5p for Targeted Therapy in Colorectal Cancer. Molecular Pharmaceutics 2019 16 (11), 4696-4710 DOI: 10.1021/acs.molpharmaceut.9b00867
DiR were purchased from Shanghai Maokang Biotech (Shanghai, China).

[3] Zhang, Y., Deng, W., Wang, W. et al. MicroRNA-206 down-regulated human umbilical cord mesenchymal stem cells alleviate cognitive decline in D-galactose-induced aging mice. Cell Death Discov. 8, 304 (2022). https://doi.org/10.1038/s41420-022-01097-z

When it reaches 80–90%, cells were stained with DIR Iodide (DiIC18) (Maokangbio, Shanghai, China) at 37 °C for 20 min according to the protocol of the manufacture

— —Written/Edited by V. Shallan【版權歸MKBio懋康所有】

上海懋康生物科技有限公司是一家涉足于生命科學和生物技術領域研究的試劑、儀器和實驗室消耗品與實驗服務工作，主要從事細胞生物學、植物學、分子生物學、免疫學、生物化學、蛋白組學。生物制藥與診斷試劑研發生產等領域。 本公司秉承“以人為本，以誠為信、合同守信”的經營理念。堅持"品質保障"的原則為廣大客戶提供優質產品。