I'm culturing adherent cells (5 x 10^5 peritoneal macrophages and bone marrow-derived macrophages), stimulating them with some drugs and a pro-inflammatory stimulus (LPS) for 24hours. I normally make a stock of 1mg/ml LPS and then use at a concentration of 10mcg/ml but I'm not seeing any increase in ROS.
After treatments I washed the cells with PBS to remove extra cell medium and trypsinized them. I centrifugated and ressuspended the cells in 10uM of H2DCFDA diluted in PBS and incubated for 30min. After the incubation period I centrifuged to remove the dye and resuspended in 100uL of PBS and I plated the cells in black 96-wells plate to read the fluorescence in a microplate fluorescence reader (488 - 535nm).
This protocol does not work fine because ROS levels was the same for all treatments, including when I used NAC as a ROS scavenger.
Usually in such cases, I add DCF-DA approximately 2 hours before termination of culture. Also, 24 hours seems to be a pretty long time to check for intracellular ROS. Did you try earlier time points? In our lab, we check for ROS in LPS stimulated whole blood. It starts appearing as early as 30 minutes. For small time points, we usually pre-incubate the cells with DCF-DA for 1 hour followed by stimulation. This is because ROS is very unstable so the dye has to be inside when your cells start making ROS. Finally, I did not understand how you can measure intracellular ROS in a microplate reader. Maybe I am missing something in what you said but usually I use flow cytometry or confocal imaging.
I hope this helps.
Good luck.
Its better to incubate your cells with dye before treatment. For this, after your cells are attached and ready for treatment, incubate the cells with dye
( do not trypsinize) for 30 mins or 1 hour (optimize time period), followed by washing with PBS, now go for your treatment, and take reading in plate reader after every 6 hours.
Hey Allan, Our protocol looks at follows:
if you like please have a look at our paper, which is mainly about ROS production in macrophages:
Article Mitochondrial reactive oxygen species enable proinflammatory...
The basic protocol looks as follows: 1) Centrifuge your cells (we seed them in normal black 96er culture plates) 2) Wash them once with HBSS (to remove medium) 3) Add your DCF/HBSS staining solution (10 µM works well in our hands) 4) Incubate at 37 °C for at least 15 and for maximal 30 minutes in an incubator 5) Remove the DCF solution and wash one time with HBSS 6) Add stimuli of your choice (PMA as a postive control works good)7) Measure in a pre-heated plate reader for 1-2 hours
I hope that helps you. Please fell free to ask any further questions.
All the best and stay healthy, Marc
I agree with the previous replies, you should check ROS after 30 minutes to one hour and sometimes even earlier after your treatment with LPS, personaly I had to make some time course experiments and I had my peek of ROS secretion around 1 hr. Also my dose of H2CDFDA is was 1uM. I hope it might help you.
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