I´m currently working with primary glioma cells cultured in DMEM suppl. with 15% FCS grown adherently and I´m trying to establish a ROS staining with DCFDA (from santa cruz). As I would like to investigate ROS production in these cells while monitoring their morphology, I chose to do so with confocal microscopy.
Unfortunately when cells are incubated in DCFA with the lowest recommended concentration of 2,5 µM for 10 min at 37°C 5% CO2, they round up and loose any adhesion to the slide. I tried to grow them on collagen coated slides which was unsuccessful as well.
A few cells pre-treated with NAC were still attached to the slide while the untreated controls were entirely detached.
Thank you for all your comments and suggestions
What is your solvent and what is the final concentration in the medium?
Hi Jordan,
DCFDA is solved in DMSO and I use a final concentration of 2,5 uM - 5 uM
Hi - it does sound as if your cells are dying (rounding up and detachment are classical signs of adherent cell death in fact toxicology devices such as the RT-CES are based upon this).
Have you control cells for the staining (cells with just DMSO added to the same concentration as your stain)? If not I would add these as it may be something strange with the DMSO you dissolved the probe in. I would also have a third slide of cells to which I add nothing but goes through the same procedure etc to see if it is an environmental problem (i.e. cells die when removed from incubator too long).
Also at what concentration is the probe? I assume you are using a 1000x stock (1mg/ml) and are adding at 1ul per ml of media? This level of DMSO should not cause a problem.
unfortunately unexpected problems with experiments is all "par for the course" in science. All we can do is try to dissect the reason.....
Actually I may have an explanation - something similar happened to me a few months ago when I was growing some neuronal cells on slides.
Have you coated the slides yourself or are they commercial?
I coated some slides myself with Ornithine & Laminin but I think the Laminin only loosely coated the glass and after a few days cells began to detach and die- I think because the Laminin itself was coming away from the glass (cells "tug" at the ECM they are on which may be the reason it does not happen immediately).
Next time you transfer the cells to the Collagen glass slide you might also put some on plastic (such as a well of a 12 well plate) - treat both with your compound and see if this effect also happens to the ones growing on plastic.
Sorry I can't be more help,
Gary
Hi Gary,
I do totaly agree with you that the cells seem to be dead and that rather quickly after incubating them with the dye. Most publications on DCFDA dye have been done on cell lines and furthermore the analysis was made mostly made by flow cytometry, where you do not see any morphological changes. I actually first grew them in 12 well plates on plastic in order to analyze them in the cytometer but here again, I did not need to harvest them as they were already detached.
Obviously DCFDA is toxic to these cells (actually to the two different primary cells I tested) and it seems that N-Acteyl cystein protects them against detachement and so against toxicity arguing for the fact that the dye is a potential source of oxidative stress itself. Nervermind I could not find any paper on that topic, some groups tried to stain the cells when there are fixed (e.g. in methanol) so I might experiment it that way.
Thanks again for your advice
Hi I would like to know what is the final concentration of DMSO that you use with DCF in your cells?
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