I am trying to work out DCFH-DA ROS assay in RAW 264.7 cells and it is giving me hard time to work it out. I am doing treatment for 24 hours and I tried both preloading and loading DCFH-DA at the end of the exposure as well. Can anyone please help. I am not sure what I am doing wrong this time as I have done this assay 2 years ago and it worked very well at that time.
Thanks
Hi Lokesh,
In my experience the DCF assay gave the best signal to noise when the cells were washed and the fluorescence readings taken in HBSS or PBS. The vitamins in DMEM and other complete media can be autofluorescent and also quench some fluorescence signals, causing poor signal/noise. I also only needed to incubate the cells with the oxidants for 2 - 4 hours to see a good signal (also not good to keep in HBSS too long as they can die, but you can keep in normal media and exchange to HBSS before the read). Are you using a positive control (e.g. H2O2, Sin1, Cu+ascorbate)?
hi lokesh,
do you really need to stimulate for 24hs? if so, i recommend you to load the dye at the end of the experiment. i would start doing a kinetic experiment with your stimulator. some stimuli induces ros generation as fast as 10min. use pma 100nM for 10-30min as a positive control. it always works.
if you are using facs: you can make stimulations with serum and any cell medium such as rpmi and dmem but it is important to load the cells with the dye in the absence of serum. i use rpmi and it works fine. after loading, wash the cells and ressuspend them in pbs.
if you are working with a fluorometer: preload the cells with the dye and work with mediums without phenol red.
good luck!!!
Thanks Adam and Fabiannno for your answers. I think both of you are pointing at important drawback in my protocol. I have been using media containing 10% FBS in the entire assay even while lysing the cells I used to add 2x lysis buffer in serum containing media. I will work on your suggestions and will let you know soon about the results. Thanks a lot again to both of you.
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