I'm using the H2DCFDA probe to measure the intracellular ROS after a certain treatment. However, every time I measured the fluorescence intensity (with flow cytometer), my untreated samples are all 'ROS positive' (high fluorescence). So high even that it's not possible to distinguish my untreated controls from my positive controls (50µM H2O2, incubated for 1 hour). I use A375 melanoma cells. I checked autofluorescence (measured fluorescence without loading DCFDA) and this was fine.
The concentration of the probe I use is 1 µM.
Does someone have an idea what is causing the high fluorescence? Any suggestions on lowering the fluorescence of my untreated samples?
Many thanks in advance!
I think you should try different doses of the probe and incubate for different time periods. Alternatively, you can measure fluorescence using plate reader.
H2DCFDA is not a specific ROS probe. It can be converted by various cellular components that are not strictly related to oxidative stress. Please refer to the attached papers for more details. For example, Amplex Red or MitoSOX are better ROS probes.
Regards, Christian
Article What does the commonly used DCF test for oxidative stress really show?
Article Measurement of Reactive Oxygen Species, Reactive Nitrogen Sp...
How long do you incubate cells with DCF? May be it's to long and cells uptake too much dye. In our lab we incubate kideny slices in 10 µM DCF-DA for 10 min.
Hi Stanislovas. I incubated for 30-40min. But maybe it is indeed too long. I'll try a shorter incubation time.
I think you should try to incubate cells with DCF at 37 °C for 10-15 minutes and wash out cells to exclude extracellular DCFH2-DA molecules before measurements.
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