DiR Iodide (DiIC18(7)) 細胞膜深紅(hóng)色熒光(guāng)探針

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搜索關鍵詞：

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

訂購信息：

産品描述：

DiI, DiO, DiD和DiR作為(wèi)一(yī)類長(cháng)鏈的親脂性二烷基碳菁類染料（Dialkylcarbocyanines）熒光(guāng)染料家族，用于标記細胞膜以及其他脂溶性生(shēng)物(wù)結構。作為(wèi)一(yī)類環境敏感型熒光(guāng)染料，當它們與膜結合或者與親脂性生(shēng)物(wù)分子（例如蛋白(bái)質，雖然在水(shuǐ)中其熒光(guāng)強度很弱）結合時，其熒光(guāng)強度顯著增強。一(yī)旦進入細胞後，它們在細胞内質膜中逐步擴散，于最佳濃度條件(jiàn)下(xià)可将整個(gè)細胞均勻染色。這些染料具很高(gāo)的淬滅系數，偏光(guāng)依賴性和很短的激發壽命。

四種染料呈現不同的熒光(guāng)顔色，DiI（橙色熒光(guāng)）、DiO（綠色熒光(guāng)）、DiD（紅(hóng)色熒光(guāng)）、DiR（深紅(hóng)色熒光(guāng)），為(wèi)活細胞多(duō)色彩熒光(guāng)成像分析和流式細胞術(shù)提供了一(yī)種便捷的工(gōng)具。DiO和DiI分别用标準FITC和TRITC濾光(guāng)器(qì)檢測，可結合使用。DiD可被633 nm氦-氖（He-Ne）激光(guāng)激發，具有比DiI更長(cháng)的激發和發射光(guāng)波長(cháng)，特别适合用于标記具本底熒光(guāng)的細胞和組織。而，DiR在體内活體成像或者示蹤中意義非凡，因其所發射的紅(hóng)外光(guāng)可以高(gāo)效地穿過細胞和組織，并且在紅(hóng)外光(guāng)範圍内，其本底熒光(guāng)水(shuǐ)平很低(dī)。

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%，适用于熒光(guāng)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(guāng)器(qì)：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(guāng)幹燥保存，有效期二年(nián)。

運輸：冰袋運輸。

應用示例（來自(zì)文獻）

文獻來源：Eisenblätter 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.

标記方法（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(cháng)位于近紅(hóng)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）來确定标記效率。後續實驗中，單核細胞（1 × 105/mL培養基）加入2ul DiR标記液（終濃度為(wèi)19.7uM）孵育5min，PBS清洗3次後，重懸于細胞培養液中。流式細胞儀檢測标記效率。

Cutaneous Granuloma (CG) Model（皮膚肉芽腫體内模型）：在小(xiǎo)鼠側區皮下(xià)注射1ml 聚丙烯酰胺凝膠（PAG）誘導皮膚肉芽腫形成。為(wèi)了誘導炎症，向PAG内加入脂多(duō)糖LPS（10ug LPS/ml）。在凝膠移植前24h靜(jìng)脈注射DiR标記的巨噬細胞或對照(zhào)巨噬細胞。使用FRI和FMT在注射後每日觀察小(xiǎo)鼠，持續7天。

FIG 1. FMT of mice showed distribution of Mϕs inside pellets. (A) FMT was performed 72 h after injection of labeled Mϕs (5 × 106) to resolve 3-dimensional distribution of Mϕs in target tissue (left, NIRF image; right, color-encoded FMT). FMT confirmed FRI results and showed that Mϕs 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标記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.

體内實時近外熒光(guāng)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(shì)項

1）熒光(guāng)染料均存在淬滅問題，請盡量注意避光(guāng)，以減緩熒光(guāng)淬滅。

2）為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

使用方法

【注意】以下(xià)使用方法僅用作參考，可根據具體的實驗條件(jiàn)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(shuǐ)DMSO中，充分溶解，即得到(dào)5mM的儲存液。【注意】未使用的儲存液分裝儲存在-20℃，避免反複凍融，且要避光(guāng)保存。

2）工(gōng)作液制備：用合适的緩沖液（如：無血清培養基，HBSS或PBS）稀釋儲存液，調整到(dào)1～5 μM的工(gōng)作濃度。【注意】工(gōng)作液最終濃度需要根據不同細胞系和實驗體系來優化。建議從(cóng)推薦濃度開(kāi)始，以10倍範圍為(wèi)區間進行最優濃度的摸索。

2. 懸浮細胞染色

1）加入适當體積的染色工(gōng)作液重懸細胞，使其密度為(wèi)1×106/mL。

2）37℃孵育細胞2～20min，不同的細胞最佳培養時間不同。可以20min作為(wèi)起始孵育時間，之後優化以保證得到(dào)均一(yī)化的标記結果。

3）孵育結束，按1000～1500 rpm離心5min。

4）去除上(shàng)清液，之後輕柔加入37℃預熱的生(shēng)長(cháng)培養液重懸細胞。

5）再重複3），4）步驟兩次。

3. 貼壁細胞染色

1）将貼壁細胞培養于無菌蓋玻片上(shàng)。

2）從(cóng)培養基中移走蓋玻片，濾掉過量培養液，将蓋玻片放(fàng)在潮濕的小(xiǎo)室内。

3）在蓋玻片的一(yī)角加入100μL的染色工(gōng)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。可以20min作為(wèi)起始孵育時間，之後優化以保證得到(dào)均一(yī)化的标記結果。

5）吸掉染色工(gōng)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，然後吸走培養基。

4. 顯微鏡檢測

1）DiD，DiO，DiI，DiR和DiS濾光(guāng)器(qì)的選擇參見(jiàn)下(xià)表：

2）多(duō)色染料的同時檢測，濾光(guāng)器(qì)按照(zhào)以下(xià)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染色的細胞分别用流式細胞儀的FL1，FL2，FL3或FL4通(tōng)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】

上海夢澤生物科技有限公司是一(yī)家涉足于生(shēng)命科學和生(shēng)物(wù)技(jì)術(shù)領域研究的試劑、儀器(qì)和實驗室消耗品與實驗服務工(gōng)作，主要從(cóng)事(shì)細胞生(shēng)物(wù)學、植物(wù)學、分子生(shēng)物(wù)學、免疫學、生(shēng)物(wù)化學、蛋白(bái)組學。生(shēng)物(wù)制藥與診斷試劑研發生(shēng)産等領域。 本公司秉承“以人為(wèi)本，以誠為(wèi)信、合同守信”的經營理念。堅持"品質保障"的原則為(wèi)廣大客戶提供優質産品。