DiI (DiIC18(3)) 細胞膜橙紅(hóng)色熒光(guāng)探針（神經元示蹤研究）

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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。其中，DiI及其衍生(shēng)物(wù)由于極其低(dī)的細胞毒性，應用最為(wèi)廣泛。不僅普遍用于活體和固定組織及細胞的神經元逆行性和順行性示蹤分析，還(hái)可以用于檢測細胞的融合和粘附，檢測發育或移植過程的細胞遷移，通(tōng)過FRAP（熒光(guāng)光(guāng)漂白(bái)恢複技(jì)術(shù)）檢測脂質在細胞膜上(shàng)的擴散過程，檢測細胞毒性，以及标記脂蛋白(bái)如LDL和HDL等。

本品以DiI的高(gāo)氯酸鹽形式提供，英文全名：1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate，CAS NO.41085-99-8，純度≥98%（TLC），适用于熒光(guāng)檢測研究。

DiI橙紅(hóng)色熒光(guāng)探針基本特性：

1） 同義名：DiI perchlorate; DiIC18(3);1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate;

2） CAS NO：41085-99-8

3） 分子式：C59H97ClN2O4

4） 分子量：933.87 g/mol

5）純度：≥98%（TLC）
6）溶解性：溶于DMF，DMSO，甲醇
7）外觀：紅(hóng)色至暗(àn)紅(hóng)色，紫色至深紫色粉末
8）Ex/Em：550/567 nm（in phosphate buffer/SDS pH 7.0）
9） 推薦濾光(guāng)器(qì)：Omega- XF108, XF32, Chroma-41002, 31002
10）化學結構圖：

保存與運輸方法

保存：4ºC幹燥避光(guāng)保存，有效期一(yī)年(nián)。

運輸：冰袋運輸。

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5mg DiI（Mw：933.87 g/mol）溶于1.07ml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。

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。

應用示例（來自(zì)文獻）：

文獻一(yī)、Wexler-Cohen Y et al. Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action. PLoS Pathog. 2009 Jul;5(7):e1000509.

使用方法（Cell-Cell Fusion Inhibition Assay）：

Jurkat E6-1 and Jurkat HXBc2 cells were labeled with DiI and DiD lipophilic fluorescent probes, respectively. The two cell populations were co-incubated, in a ratio of 1∶1, for 6 h in the presence of eight different concentrations of the inhibitory peptides.Prior to measurements the cells were washed, spinned, dissolved in PBS, and put on ice. Cells co-incubated without the presence of peptides served as an optimal fusion reference. Unlabeled cells that were handled similarly served as an intrinsic fluorescence control.

Cells labeled separately with DiI or DiD were used to adjust the optimal separation of fluorescent signals. Jurkat HXBc2 cells labeled with DiI were co-incubated with Jurkat HXBc2 cells labeled with DiD for a fusion background that was subtracted from the measurements of the experiment.The following alterations were applied to the original protocol: (i) 5 µL of a 1 mg/mL DiI or DiD solution in dimethylsulfoxide (DMSO) was added to 1 mL of 4×106 cells/mL Jurkat E6-1 or Jurkat HXBc2 cells, respectively.(ii) For each data point 150,000 events were collected. Measurements were performed on a FACSort machine, upgraded to a FACSCalibur cell analyzer (Becton Dickinson).

文獻二、Mazzoni M et al. Distribution, organization and innervation of gastric MALT in conventional piglet. J Anat. 2011 Nov;219(5):611-21.

使用方法（Tissue preparation and DiI tracing in fixed tissue）：

Small crystals of DiI were diluted at 3% in 100% ethanol and evaporated onto small glass beads (about 200 μm). The glass beads were placed in the middle of the gastric folliclesto detect if neurones projecting to the follicles were present. In other samples, after gently removing the overlying epithelium by the use of entomological forceps, glass beads were inserted into the lamina propria to identify whether neurones projecting to the mucosal layers were present.

Fig 1. Tangential sections of pig gastric mucosa at the level of the diverticulum.(A) Stereomicroscopy showing DiI‐coated beads (arrow) applied to a follicle in fixed tissue after 8 months of incubation. (B,C,E) DiI crystals applied to the follicles; note that the tracer was homogeneously distributed inside the follicles. (B) Labelled neurones (arrowhead) and processes entering the labelled follicle. (C) Polyhedral‐labelled neurone (arrowhead) more than 400 μm from a labelled follicle. (D) Higher magnification of the neurone shown in (C). (E) Elongated neurone (about 400 μm from the labelled follicle) along a thin nervous fascicle (arrowhead). (F) Higher magnification of the neurone shown in (E). (G,H) Two labelled neurones with an ovoid shape. Note that the neurones showed smoothly contoured soma and an eccentrically located nucleus (D,G,H,)

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

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