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DiOC2(3) (3,3'-Diethyloxacarbocyanine Iodide) - FAQs

查看更多产品信息 DiOC2(3) (3,3'-Diethyloxacarbocyanine Iodide) - FAQs (D14730)

14 个常见问题解答

当我使用膜电位指示剂时，看到神经元周围出现了较高的背景，如何降低背景干扰？

如果使用我们的FluoVolt 膜电势试剂盒（货号F10488），该试剂盒包含一种背景抑制剂，可改善这一问题。对于其他指标剂，可以考虑使用BackDrop 背景抑制剂（货号R37603、B10511和B10512）。

我用DiI一类的亲脂性花青染料进行细胞染色，但是当我试图继续用抗体标记时信号却丢失了。 这是什么原因所致？

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。

快反应膜电位探针和慢反应膜电位探针有什么区别？

在周围电场的作用下结构变化的分子可用作检测瞬时（毫秒级）电位变化的快反应探针。慢反应染料则会进入去极化细胞，结合蛋白或膜。增强去极化会造成额外的染料流入，增强荧光强度；过极化的特征则是荧光强度下降。快反应探针通常用于完整心脏组织的电位活动成像，或测量药理刺激引起的膜电位变化。慢反应探针常用于探索线粒体功能和细胞活力。

你们提供哪些类型的膜电位指示剂？我该如何根据自己的试验选择？

膜电位指示剂选择指南请见此处（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/ion-indicators/membrane-potential-indicators.html）。

亲脂性示踪剂跨膜转运需要多久？FAST亲脂性染料能快多少？

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。

我应该使用哪种亲脂性示踪剂（DiO、DiI、DiD等）？

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。

我想标记两种细胞群体然后进行细胞融合实验，哪种试剂最适合来成像？

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）。

I am seeing high background outside of my neuronal cells when using membrane potential indicators. What can I do to reduce background?

If you use our FluoVolt Membrane Potential Kit (Cat. No. F10488), the kit provides a background suppressor to reduce this problem. For other indicators, consider the use of BackDrop Background Suppressor (Cat no. R37603, B10511, and B10512).

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

I stained my cells with a lipophilic cyanine dye, like DiI, but the signal was lost when I tried to follow up with antibody labeling. Why?

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.

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

What is the difference between fast and slow-response membrane potential probes?

Molecules that change their structure in response to the surrounding electric field can function as fast-response probes for the detection of transient (millisecond) potential changes. Slow-response dyes function by entering depolarized cells and binding to proteins or membranes. Increased depolarization results in additional dye influx and an increase in fluorescence, while hyperpolarization is indicated by a decrease in fluorescence. Fast-response probes are commonly used to image electrical activity from intact heart tissues or measure membrane potential changes in response to pharmacological stimuli. Slow-responding probes are often used to explore mitochondrial function and cell viability.

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

What type of membrane potential indicators do you offer and how should I choose one for my experiment?

A membrane potential indicator selection guide can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/ion-indicators/membrane-potential-indicators.html).

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

How long does it take for lipophlic tracers to transport along the membrane? How much faster are the FAST lipophilic dyes?

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%.

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

Which form of the lipophilic tracers (DiO, DiI, DiD, etc) should I use?

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.

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

I want to label two cell populations and then perform a cell fusion assay. Which reagents are best for imaging this?

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).

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