一、百螢 橙色熒光示蹤探針Cyto Trace CMTMR簡(jiǎn)介

CytoTrace 橙色CMTMR在化學(xué)上與CellTracker 橙色CMTMR相同。CytoTrace Orange CMTMR可以自由地穿過細(xì)胞膜進(jìn)入細(xì)胞，然后轉(zhuǎn)化為不滲透細(xì)胞膜的反應(yīng)產(chǎn)物。CytoTrace Orange CMTMR染料可以在活細(xì)胞中保留數(shù)代。染料轉(zhuǎn)移到子細(xì)胞中，但不轉(zhuǎn)移到種群中的相鄰細(xì)胞中。CytoTrace 橙色CMTMR染料旨在顯示至少72小時(shí)的熒光，并且該染料具有理想的跟蹤特性。CMTMR染料穩(wěn)定，在細(xì)胞中保留良好，并且在生理pH下發(fā)出明亮的熒光。此外，CytoTrace 橙色CMTMR染料的激發(fā)和發(fā)射光譜與GFP（綠色熒光蛋白）已很好地分離，并且可以進(jìn)行多重光譜。

二、百螢 橙色熒光示蹤探針Cyto Trace CMTMR實(shí)驗(yàn)方案

該協(xié)議僅提供指南，應(yīng)根據(jù)您的特定需求進(jìn)行修改。

1.準(zhǔn)備2-10 mMDMSO儲(chǔ)備溶液

對(duì)于＃22014添加45微升DMSO成50?μ?克小瓶使2mM的儲(chǔ)備溶液（1毫克/毫升，相當(dāng)于1.8毫摩爾）;

對(duì)于＃22015添加36微升DMSO成50?μ?克小瓶制成10mM儲(chǔ)備溶液（1毫克/毫升，相當(dāng)于1.46毫摩爾）;

對(duì)于＃22016，添加153 uL ml DMSO以制成10 mM儲(chǔ)備溶液（1 mg / ml相當(dāng)于1.53 mM）；

對(duì)于＃22017，添加215μLDMSO以制成10 mM儲(chǔ)備溶液（1 mg / ml相當(dāng)于2.15 mM）;

對(duì)于＃22020，將4.2 mg溶于1 ml DMSO中制成10 mM儲(chǔ)備溶液（1 mg / ml相當(dāng)于?2.4 mM）；

注意：儲(chǔ)備溶液應(yīng)及時(shí)使用；應(yīng)將所有剩余的溶液等分并在<?-20?o?C下冷凍。避免重復(fù)凍融循環(huán)，并避光

2.準(zhǔn)備染料工作液

在使用前，通過用Hanks和20 mM Hepes緩沖液（HHBS）或您選擇的pH 7緩沖液稀釋步驟1中的DMSO儲(chǔ)備溶液，準(zhǔn)備好1至20?μM的染料工作溶液。

3.用流式細(xì)胞儀或熒光顯微鏡分析細(xì)胞：

3.1用測(cè)試化合物處理細(xì)胞所需的時(shí)間。

3.2離心細(xì)胞，每管得到2-10 x105細(xì)胞。

3.3將細(xì)胞重懸于500 μL?的染料工作溶液中（來自步驟2）。

3.4將細(xì)胞與染料溶液在室溫或37°?C下避光放置15至30分鐘。

3.5從細(xì)胞中除去染料工作溶液，用HHBS或您選擇的緩沖液洗滌細(xì)胞。將細(xì)胞重懸于500 μL預(yù)熱的HHBS或培養(yǎng)基中，每管可得到2-10 x 10?5個(gè)細(xì)胞。

3.6用流式細(xì)胞儀（FL1通道）?或熒光顯微鏡監(jiān)測(cè)Ex / Em = 490/520 nm處的熒光變化。

注意：對(duì)于細(xì)菌細(xì)胞染色：將母液在通過測(cè)試細(xì)菌過夜生長(zhǎng)而預(yù)先調(diào)節(jié)的營(yíng)養(yǎng)肉湯中以1：800的比例稀釋時(shí)，染色是有效的，但是也可以使用新鮮的營(yíng)養(yǎng)肉湯或PBS。細(xì)菌懸浮液應(yīng)用PBS稀釋至105?- 10?7每毫升。將1 ml溶液加到.45 μm過濾器（25mm）上并真空過濾除去溶液，然后加入1ml染料溶液并在室溫下孵育5-10分鐘，即可對(duì)細(xì)菌染色。

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三、百螢 橙色熒光示蹤探針Cyto Trace CMTMR參考文獻(xiàn)

Fluorescence-Based Transport Assays Revisited in a Human Renal Proximal Tubule Cell Line
Authors:?Caetano-Pinto P, Janssen MJ, Gijzen L, Verscheijden L, Wilmer MJ, Masereeuw R.
Journal:?Mol Pharm (2016): 933

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance
Authors:?Manna A, De Sarkar S, De S, Bauri AK, Chattopadhyay S, Chatterjee M.
Journal:?Phytomedicine (2015): 713

Cell membrane tracker based on restriction of intramolecular rotation
Authors:?Zhang C, Jin S, Yang K, Xue X, Li Z, Jiang Y, Chen WQ, Dai L, Zou G, Liang XJ.
Journal:?ACS Appl Mater Interfaces (2014): 8971

A multiple model probability hypothesis density tracker for time-lapse cell microscopy sequences
Authors:?Rezatofighi SH, Gould S, Vo BN, Mele K, Hughes WE, Hartley R.
Journal:?Inf Process Med Imaging (2013): 110

Evaluation of stability and sensitivity of cell fluorescent labels when used for cell migration
Authors:?Beem E, Segal MS.
Journal:?J Fluoresc (2013): 975

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies
Authors:?Klein J, Leupold S, Biegler I, Biedendieck R, Munch R, Jahn D.
Journal:?Bioinformatics (2012): 2276

Horizontal DNA transfer from donor to host cells as an alternative mechanism of epithelial chimerism after allogeneic hematopoietic cell transplantation
Authors:?Waterhouse M, Themeli M, Bertz H, Zoumbos N, Finke J, Spyridonidis A.
Journal:?Biol Blood Marrow Transplant (2011): 319

The exocytosis of fluorescent nanodiamond and its use as a long-term cell tracker
Authors:?Fang CY, Vaijayanthimala V, Cheng CA, Yeh SH, Chang CF, Li CL, Chang HC.
Journal:?Small (2011): 3363

The interplay between Leishmania promastigotes and human Natural Killer cells in vitro leads to direct lysis of Leishmania by NK cells and modulation of NK cell activity by Leishmania promastigotes
Authors:?Lieke T, Nylen S, Eidsmo L, Schmetz C, Berg L, Akuffo H.
Journal:?Parasitology (2011): 1898

Cell electrofusion visualized with fluorescence microscopy
Authors:?Trontelj K, Usaj M, Miklavcic D.
Journal:?J Vis Exp. (2010)