DCFH-DA is an organic molecule that is poorly soluble in water. The best approach is to prepare a concentrated stock in dry, cell culture grade DMSO, for example 10 mg/mL, aliquote and freeze. Then dilute to 1 uM or so in buffer for loading. You will need to determine the optimal loading time and dye concentration.
After loading, allow the cells to recover in media so the DA group can be cleaved and you're ready to go with your assay.
Be aware that there are many caveats to the various fluorescent ROS indicators, including DCFH. I recommend looking at some papers by Dr. Ischiropoulos
https://www.researchgate.net/profile/Harry_Ischiropoulos/
For example, below is an excellent review:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911769/
i want to investigate whether pre-treatment with my fruit extracts can hinder ROS accumulation in my cells. My protocol is:
1. seed cells, allow adhesion 24hr
2. wash & treat cells with fruit extract 24hr
3. add H202 / AAPH (as inducers of oxdiative stress) for 1 hour
4. wash & stain with DCFH-DA (final conc. 10uM in PBS) for 30min
5. read signal intensity.
Does this sound alright?
I would reverse #3 and #4.
You need the cells to load with and then convert the non-active DCFH-DA to the active DCFH (which is fairly cell impermeant, so once loaded it will stay inside), which can then be oxidized to DCF which fluoresces. Once thusly primed, treat the cells with your inducing agents and see what you get.
Be aware that DCFH does not react with H2O2 directly, the chemistry is quite complex, I really recommend reading the review I linked above.
Thanks for your advice, much appreciated. I'll take a closer look at the review.
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