DiO (DiOC₁₈(3)) 細胞膜綠色熒光(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

DiO訂購信息：

DiO綠色熒光(guāng)探針基本特性：

1. 化學名：Benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-, perchlorate

2. 同義名：DiO perchlorate; DiOC18(3); 3,3-Dioctadecyloxacarbocyanine perchlorate;

3. 推薦濾光(guāng)器(qì)：Omega-XF100, XF23, Chroma-41001, 31001

4. CAS NO：34215-57-1

5. 分子式：C53H85ClN2O6

6. 分子量：881.72g/mol

7. 外觀：橙黃至橙色固體

8. 純度：≥98%

9. Ex/Em：484/501 nm（甲醇）

10. 溶解性：溶于DMF，DMSO，甲醇

11. 化學結構圖：  

應用示例（DiO，來自(zì)文獻）：

文獻一(yī)：Piriou L et al. Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species. Cytometry. 2000 Dec 1;41(4):289-97.

使用方法：

For DIOC18(3) labeling, 1 ml of complete medium containing 10⁶ cells was mixed with 10 μl of 3 mM DIOC18(3). The cells were incubated at 37°C for 15 min and rinsed three times with PBS. Cell labeling and cell viability were confirmed by flow cytometry before use in the test. Cell viability was assessed by PI, which permeates only through the membrane of dead cells and emits a red fluorescence (10 μg/ml, optimal concentration determined after a calibration assay. The test was performed when cells were labeled homogeneously and cell viability was higher than 80%.

Fig 1. FCA of target cells in an NK cell cytotoxicity assay. Four cell subpopulations are shown by fluorescent staining with DIOC18(3) (green: x axis, FL1) and PI (red: y‐axis, FL3). The fluorescence contour plot cytogram (FL1 versus FL3) shows target cells labeled with DIOC18(3) (FL1) versus the dead cells labeled with PI (FL3). Quadrant 1 = lysed effector cells; quadrant 2 = live effector cells; quadrant 3 = lysed K562 target cells; quadrant 4 = live K562 target cells.

文獻二、Romero-Pérez GA et al. Orally Administered Salacia reticulata Extract Reduces H1N1 Influenza Clinical Symptoms in Murine Lung Tissues Putatively Due to Enhanced Natural Killer Cell Activity. Front Immunol. 2016 Mar 31;7:115. 

使用方法：

YAC-1 cells were cultured in 75-cm2culture flasks containing 10 mL of culture media and incubated in a humidified chamber (37°C, 5% CO2). After 3-day incubation, YAC-1 cells were stained for 15 min at 37°C with 10μM Dioc18 [3,3′-dioctadecyloxacarbocyanine perchlorate] to cause green fluorescence of cell membranes. YAC-1 cells were then washed twice with 1mL HBSS and seeded in 100 μL of culture media in 96-well culture plates in a concentration of 1.0 × 10⁴ cells (for coculture with splenocytes) or 5.0 × 103cells (for coculture with pulmonary cells) per well. 

For the NK activity assays, YAC-1 cells were used as target cells, and splenocytes and pulmonary cells as effector cells. One hundred microliters each of splenocyte and pulmonary cell suspensions were added to each well containing DioC18-labeled YAC-1 cells to achieve 25:1 and 10:1, and 10:1 and 5:1 effector cell:target cell (E:T) ratios, respectively. After incubation for 4 h, 10 μL of ethidium monoazide bromide (EMA, 0.5 ng/mL) solution was added to each well for nuclear staining of dead cells. The ethidium was bound to nuclei by photo affinity using a 26-W fluorescent light (5 cm distance) for 10 min at room temperature. After washing twice with PBS containing 0.5% BSA, the cells were fixed with 2% PFA overnight. Each sample was incubated in duplicate and analyzed using an Accuri C6 flow cytometer.

Fig 2. Representative images of flow-cytometric analysis for the determination of NK activity. To discriminate the target cells, live gate (red line) was set in the FL1 histogram on the green fluorescent (A,B). The target cells were further analyzed with a dot plot of FL1 and FL3 (C–E). 

傳統細胞膜探針：

1. DiI, DiO, DiD和DiR

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，尤其是DiI，廣泛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。

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，最佳濃度條件(jiàn)下(xià)可将整個(gè)細胞染色。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)。

2. DiA

DiA為(wèi)親脂性氨基苯乙烯基染料（dialkylaminostyryl dye），常用于神經元的示蹤，可與DiI聯合使用進行神經細胞的雙色标記。研究發現，某些情況下(xià)當DiO不能(néng)标記固定組織時，DiA可得到(dào)良好标記效果。DiA與磷脂雙層膜結合後，光(guāng)譜遷移明顯加強。正由其發射光(guāng)譜非常廣，在适當的濾光(guāng)片下(xià)，可以綠色、橙色，甚至紅(hóng)色熒光(guāng)被檢測到(dào)。

産品訂購信息：

細胞膜探針光(guāng)譜和檢測濾片：

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

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