DiD' oil; DiIC₁₈(5) oil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate) - FAQs

由于这些染料插入脂质膜，任何对膜的破坏都将导致染料流失。这包括用Triton X-100之类的去垢剂或是甲醇之类的有机溶剂通透。通透是胞内抗体标记所必需的，但它会导致染料流失。相反，CFDA SE之类活性染料能够共价连接到细胞组分，因此可以在固定和通透之后更好的保留。

转运过程相当缓慢：在活体组织中大约6 mm/天，在固定组织中更慢。亲脂性羰花青示踪剂从开始给样处扩散到神经元末端，可能需要数天甚至数周的时间。FAST DIO和DiI类似物（具有不饱和烷基尾）可以提高50％左右的转运速度。

请选择与您现有的激发光源和发射滤光片组/通道兼容的染料。固体、糊和晶体形式的染料可直接施加到组织中的神经元。如需标记培养的细胞或进行显微注射，可采用溶液或固态形式的亲脂性染料。

亲脂性花青染料非常适合此类测定，因为它们会掺入到细胞膜中，随着细胞融合之后共用细胞膜，染料也随之共享。例如，一个细胞群体可以用DiI（橙红色）进行标记，而另一细胞群可以用DIO（绿色）进行标记，当细胞融合时合并的颜色为黄色（使用双带通滤光片成像）。

这是不推荐的。这些染料与DNA和RNA的结合会影响核酸的正常功能，扰乱转录和增殖。诸如CellTracker染料或Qtracker试剂在不严重扰乱细胞正常活动的条件下对其进行追踪。如果您仍需要使用核酸染料进行标记且细胞是哺乳动物和非血液来源的话，CellLight 细胞核试剂可通过瞬时转染进入细胞，在核表达蛋白上表达GFP或RFP长达数天而不影响其功能。

请浏览这里（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html）以帮助您选择适合您的应用的产品。首先确定追踪细胞的时间，然后考虑染料结合机制。钙黄绿素染料标记均匀且对短期细胞迁移示踪效果极佳，但也会被某些类型细胞迅速外排。亲脂性的花青素染料，如DiI，DIO，和类似的染料能标记细胞膜而不破坏其功能，并且能持续更长时间，但如果发生膜融合则可能会染上其他细胞。此外，它们还会在透化过程中丢失。CellTracker染料更有利于长期标记，其带有温和的氯甲基反应基团使之能够与细胞组分共价结合。CFDA SE也能共价地结合于细胞组分。在所有列出的试剂中，细胞内保留与否取决于细胞分裂的速率和细胞的固有特性（主动外排，膜和蛋白质的周转率等）。其中共价结合试剂比非共价结合的试剂展现出更长的保留时间。

Qtracker试剂是最持久并且荧光强度最高的细胞示踪染料，它通过内吞作用被细胞摄入。在许多样品中它们产生的信号可以持续检测长达数周，而且信号足够强，即使在固定和通透甚至加热和石蜡处理过程中，仍然能够维持较好的荧光信号。

This is expected. DiD (which is far-red fluorescent) is never as uniform as DiI (which is orange fluorescent). If uniformity is desired, try increasing the label time and concentration, but it still isn't likely to be as uniform as DiI. CellMask Deep Red Plasma Membrane stain is much more uniform and is about the same wavelength as DiD. However, if you intend to do cell tracking over days, CellMask stain has not been tried for that application.

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Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

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The transport is fairly slow, around 6 mm/day in live tissue and slower in fixed tissue, so diffusion of lipophilic carbocyanine tracers from the point of their application to the terminus of a neuron can take several days to weeks The FAST DiO and DiI analogs (which have unsaturated alkyl tails) can improve transport rate by around 50%.

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Select the dye that is compatible with your available excitation source(s) and emission filter set/channels. The solid, paste and crystal forms can be applied directly to neurons in tissues. For labeling cells in culture or microinjection, the lipophilic dyes in solution or solid form can be used.

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Lipophilic cyanine dyes are preferred for this sort of assay, since they insert into cellular membranes and then, upon fusion, are shared by the fused cells as the membranes are shared. For example, one cell population can be labeled with DiI (orange-red) and another cell population can be labeled with DiO (green), and when the cells fuse, the combined color appears yellow (when imaged with a dual-bandpass filter set).

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This is not recommended. When these stains bind to DNA and RNA, they may affect the normal function of the nucleic acids, disrupting transcription, as well as replication. Other reagents, such as CellTracker dyes or Qtracker reagents are more optimized for tracking without disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-hematopoietic, CellLight nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

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Please see this Web link (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html) to help you choose the right option for your application. Start by planning how long you want to track your cells, then consider the mechanism of binding. Calcein dyes are very uniform in label and are good for short-term cell migration, but may be rapidly effluxed from some cell types. Lipophilic cyanine dyes, such as DiI, DiO, and similar dyes label cell membranes, don’t disrupt function, and can last longer, but have the potential to cross to other cells if membranes fuse. They are also lost upon permeabilization. CellTracker dyes are better for longer-term labeling, as they possess a mildly reactive chloromethyl moiety that allows covalent binding to cellular components. CFDA SE also covalently binds to cellular components. With all the reagents, their retention within cells is dependent upon the rate of cell division and the inherent properties of the cell (active efflux, membrane and protein turnover rates, etc.) and reagents that allow for covalent attachment exhibit longer retention than those that do not.

The longest-lasting and brightest options are the Qtracker reagents, which are taken up through endocytosis. These are so bright individual quantum dots can be detected, and are also robust enough to survive not only fixation and permeabilization, but even the heat and solvents used in paraffin processing.

Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.