I have been trying to do this ROS assay with SH-SY5Y cells but the readout is incredibly poor with little to no variation even when spiked with H2O2 if anyone has done this experiment with these cells I would appreciate a protocol!
I'm looking at an extracts ability to scavenge free radicals much like gallic acid or quercetin . We induce the damage to the cells with rotenone.
Which dye are you using to detect ROS. and how are you detecting the fluorescence?
On which kind of ROS are you exactly interested? Please give more details about the protocol used, dye, how do you measure, etc.
DPPH assay, and DCF DA staining can give you insights of ROS
Ive been using DCFH-DA dye and inducing ROS with H2O2 as a control, and rotenone. The dye I'm using a 10 uM solution of since it contains methanol in stock solution and the cells were dying due to the methanol. Im measuring ROS activity using a BioTek synergy 2 fluorescence reader at 485/528
DCF DA is soluble in DMSO as well.. If you can keep the DMSO concentration low it would not be toxic to cells.. You have not mentioned how you lyse your cells.. but you tend to lose DCF fluorescence in all the time you take to lyse the cells. If you can look at DCF stained cells under microscope.may be it will help you.
The protocol from cell bio mentioned if i use a clear black bottom plate lysing is not necessary. http://www.cellbiolabs.com/in-vitro-ros-rns-assay this is the kit that i am utilizing.
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