为什么我看不到活细胞荧光指示剂信号有明显变化？
不管何种活细胞指示剂染料（如钙指示剂，pH指示剂，金属离子指示剂），请务必确保上样过程中无血清，否则血清会过早地切割带有AM酯基的染料并非特异性结合染料。在检测样品之前，请使用阳性对照优化染料浓度和染色时间来得到最佳的信号与背景比值。做阳性对照时缓冲液一定要包含已知浓度的自由离子和用于打开离子通道的离子载体（例如Fluo-4 AM一类的钙指示剂，它包括缓冲液中加入钙与卡西霉素，又比如pH指示剂，不同pH值的缓冲液与尼日利亚霉素相结合）。类似于CellROX Green 或H2DCFDA需要一个细胞活性氧（ROS）刺激作为阳性对照，例如甲萘醌。最后，确保成像系统具有灵敏的探测器。例如相对于显微镜或流式细胞仪，读板仪检测信号和背景差异的能力就比较低。
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).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
