When I worked with DCFDA in B. pertussis, sometimes I see a yellow color after one hour of incubation (protected from light at room temperature) and sometimes not. For carboxy-DCFDA, I don't see changes of color at all, and I notice that I need more incubation time. Should I expect these changes of color? I'm using 50uM, which is a high concentration comparable to what I have found in many protocols, and if I leave the sample reacting, the fluorescence intensities increase more and more. How do I know when to stop and do the measurements?
Thank you,
Vanessa
I only use CM-H2-DCFDA to detect ROS production in eucaryote cells or red blood cells. Sorry not to be able to help you in your specific model. Best regards. Philippe Chadebech
Hi Vanessa.
DCFDA is NOT a probe for ROS detection.
As Philippe said you should try a reduced form of fluorescein, like DC-H2-FDA.
Di-hydro-ethidium (red) is also used for ROS detection.
As you work with bacteria, maybe you should try mitochondrial probes such as MitoTrackerOrange H2-CMTMRos, MitoTrackerRed H2-CMXRos, DiHydroRhodamine123 (which is a green rhodamine).
As the fluorescent form of the probe will be produced when ROS are produced, and ROS are produced continuously in time, fluorescence will accumulate in time. It is then mandatory that you measure all your samples at the same time.
When I perform ROS production measurements in plant cells by flow cytometry, I load cells with a positive, constitutive dye, for 30 minutes (here MitoTrackerGreen). I run once my sample "A -blank". I add my ROS probe and re-run immediately, "A-t0". Sample "B-blank", "B-t0" ; "C-blank", "C-t0" ...
When all the samples are run once, I re-run all the samples 90 minutes later, in the same order : "A-t90" ; "B-t90" ; "C-t90" ...
Oliv
AreHi Vanessa
I am also performing measurements with DCFDA (sigma) with rat cardiomyocytes and I have similar problems. I tried different protocols. Using PBS with glucose, DMEM without phenol red and low serum. Washing cells after treatments and before DCFDA. Adding DCFDA 2X directly to wells without washing 1h prior finishing treatments. Using 10 and 20uM and none of them worked any time. I never see differences between control and treatments (Palmitate to induce ROS and Estradiol as antioxidant). I use a microplate fluorescence reader with bottom measurement setting.
As for what you mention about the colour, I experienced the same once. It happened when I added the DCFDA 2X directly to the medium with the compounds and I incubated it for one hour. The wells with palmitate didn't turn into yellow but the control and estradiol did. I guess its due to some interaction with the treatments.
Have you used other labeling fluorophores such as MitoSox? I would like, but it's very expensive and I looking for some advice first.
Thank you all for your recommendations.
Agusti, I've only used DCFDA, DCFHDA and carboxyH2DCFDA. It seems that I'm getting better results with the carboxy form.
Dear Vanessa: Color is an extreme reaction for a fluorophore like the ones you are using. Have you tested the reactivity (fluorescence) of those probes with a chemical ROS generation system or H2O2? You might find a surprise. See Pino JA et al., Reproduction 2013.
At least H2DCFDA also reacts with RNS and only reacts with H2O2 in the presence of peroxidases.
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