我试着用活性氧（ROS）指示染料标记我的细胞，但没有观察到信号的明显差异。这是怎么回事？
首先，确保您同时拥有一个阴性（未处理）和一个阳性（ROS诱导）样本进行比较。100 µM甲萘醌1小时或是50 µM奈法唑酮24小时可以作为很好的阳性对照。H2O2 虽然不能很好地与CellROX染料兼容，但也可以用于阳性对照。某些如H2DCFDA的染料需要酯酶切割，所以不能在血清（血清中包含的酯酶可过早切割染料）存在的情况下进行培养。如果您的阳性对照相对于阴性对照没有明显变化，尝试着增加染料浓度和标记时间。我们的实验指南给出了启动建议。进行活细胞成像时尽可能得快。只有两种染料（CellROX Green和CellROX Deep Red）经甲醛固定处理后能保留在样品中。最后，确保您使用的滤光片和仪器设置与染料的激发和发射光谱相匹配。
I want to assay cells for reactive oxygen species using carboxy-H2DCFDA, but I want to do so with a plate reader instead of microscope. Will it work?
It has been done. The problem is that plate readers are less sensitive than microscopes, with far less signal-to-background difference. It is worth trying, but first optimize concentrations and loading times with control cells, use a plate with little to no autofluorescence, and possibly optimize the gain setting in order to get the best signal possible. But don't expect the same sensitivity, even with optimization.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I have GFP-transfected cells and need to label for reactive oxygen species. Can I use H2DCFDA?
This is not recommended as the two dyes overlap in the emission wavelength. There are other ROS reagents available in different wavelengths, such as CellROX Deep Red, which emits in the far-red range (665 nm), or dihydroethidium, which is emits in the visible red range (620 nm).
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I labeled my cell with CM-H2DCFDA for reactive oxygen detection, but upon illuminating the cell there is a significant increase in fluorescence in the control cells. Why?
If the cell is overloaded with dye, the high intracellular concentration of the dye may lead to dye-dye quenching. Upon illumination, photobleaching will occur, which will reduce the dye-dye quenching and actually increase the fluorescence (for a while, but then it will start decreasing). To solve the problem, reduce the concentration and incubation time, and try a range of incubation times and concentrations.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I need a formaldehyde-fixable reactive oxygen species detection assay. Is H2 DCFDA fixable?
H2DCFDA and similar derivatives are not fixable. The same goes for dihydroethidium and dihydrorhodamine. However, CellROX Deep Red and CellROX Green are retained for a limited time upon fixation with formaldehyde. CellROX Green may be retained upon subsequent Triton X-100 permeabilization. Avoid the use of any acetone or alcohol-based fixatives or fixatives that include alcohol, such as formalin.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
