CellROX™ Green Reagent, for oxidative stress detection - FAQs

许多对哺乳动物细胞使用的染料也可以用于细菌。例如，CellROX Deep Red试剂经证实可以用于检测B. subtilis的活性氧（见参考文献http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf）。如果您对某一特定染料有兴趣但不知道它对您的细菌是否有效，最佳途径是通过文献检索的方式查看它是否已经过试验。如果没有，则可能需要您亲自测试。

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。

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.

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CellROX Green dye diluted in a DMSO stock solution is stable for multiple freeze-thaw cycles, but it is not stable long-term in aqueous solution. Please note, every time the stock solution is opened, some of the dye can oxidize, which will increase background noise. DMSO readily absorbs moisture from the air, especially when cold, so be sure to completely thaw the stock solution to room temperature before opening and only open briefly. Water in the DMSO will gradually cause the dye to precipitate and come out of solution. Minimize the number of times you use a stock solution to several freeze-thaw cycles or make small aliquots to reduce the number of freeze-thaw cycles.

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The main difference between the CellROX Green Flow Cytometry Assay Kit (Cat. No. C10492) and CellROX Green Reagent, for oxidative stress detection (Cat. No. C10444), is the suggested final working concentrations and application. The CellROX Green in the CellROX Green Flow Cytometry Assay Kit is specifically designed for flow cytometry analysis and is used at a lower concentration than what is required for imaging, as fluorescence-activated cell sorting (FACS) is a very sensitive detection system.

The standalone CellROX Green Reagent (Cat. No. C10444) is marketed for fluorescent microscopy and require working concentrations appropriate for imaging. This can typically be as much as 10-fold higher than the concentration recommended for flowcytometry. The standalone CellROX Green Reagent can also be used for flowcytometry; however, the working concentration will have to be optimized. For the flow cytometry quick reference, use the link below.

CellROX Flow Cytometry Assay Kit Quick Reference

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Many dyes that are used on mammalian cells have also been shown to be useful in bacterial cells. For example, CellROX Deep Red Reagent has been shown to work in B. subtilis (see Reference: http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf). If you are interested in a particular dye, but are not sure if it will work on your bacteria, literature searches are the best way to check to see if it has been tested. If not, then it may be worth testing yourself.

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First, make sure you have both a negative (untreated) and positive (ROS-induced) sample to compare. A good positive control can be the use of 100 µM menadione for one hour or 50 µM nefazodone for 24 hours. H2O2 can also be used, though it does not work well for CellROX dyes. Some dyes, such as H2DCFDA, require esterase cleavage, so don't incubate in the presence of serum (which contains esterases that can prematurely cleave the dye). If your positive control does not show significant change compared to the negative control, try increasing the concentration and label time for the dye. Our manuals give starting recommendations. Be sure to image your live cells as soon as possible. Only two dyes (CellROX Green and CellROX Deep Red) are retained with formaldehyde fixation. Finally, make sure you are using filters and instrument settings to match the excitation and emission spectra of the dye.

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