I would like to use 2′,7′-Dichlorofluorescin diacetate to assess the ROS production in a549 cells growing in a 96-well microplate.
I have found protocols similar to the following in various papers, but I still have questions about the procedure.
- Seed 5000 a549 cells by wells in a 96-well plate, incubate at 37°C overnight for adherence.
- Preload the cells with 10 µm H2DCFDA in phenol red-free medium or in PBS for 30 min, 37°C in the dark (As I do not have a phenol-red free medium in stock, I would like to use PBS. But would it not be deleterious for the cells, or at least for their ROS metabolism, to be conserved in just PBS for such a long time?)
- Remove the medium containing H2DCFDA and replace by a medium containing the ROS inductor (in my case, I would like to use H2DCFDA to study the impact of an ROS-inductor exposition for 48h. Considering the important time of incubation (48h), does a H2DCDA-loading after the exposition would not be preferable?)
- Remove the ROS inductor containing medium and replace by 100 µl of PBS
- Read the emission on a microplate reader (Ex/Em: ∼492–495/517–527 nm)
Any suggestions or advice would be welcome.
I would suggest following:
- Measure how many cells are attached to the well. You may find many cells do not attach. Alternatively, you may measure metabolic activity per well.
- H2DCFDA will diffuse from the cells. You will need how much H2DCFDA per well as well as what is your actual reading.
- If H2DCFDA does not work, you might have to consider other dyes that easily go into the cells and do not diffuse from the cells.
- It feels 48 hrs of inducing ROS is too long for the reason above. I would suggest doing it within a day.
- Think about the direct interaction between H2DCFDA and the agent. It does not say what, so others cannot make any comment on this.
We did some stress-resistance assays using different cell types that can be applicable to other cell types. We did not use H2DCFDA.
Article Multiplex stress resistance in cells from long-lived dwarf mice
Hi Valentin,
Dr. Murakami provided you with excellent suggestions. I just would like to reiterate that incubating cells with ROS-sensitive fluorochrome(s) for prolonged periods of time (e.g. 24/48 hrs) will markedly reduce the specificity of your measurement due to auto-oxidation of the fluorochrome(s). If your cells are induced/activated to generate ROS, you should be able to detect the signal within an hour or so....You may consider treating your cells for prolonged periods of time (e.g. 48 hrs) and only then adding H2DCFDA for a few hours at the end of the experiment (at this point you could replace your cell culture medium with HBSS or PBS without jeopardizing cell viability).
One more thing to keep in mind when measuring ROS in cells grown on plastic plates: despite which fluorochrome you will choose (eg. H2DCFDA, DHR123 etc) the signal will be relatively weak if "regular" (e.g. clear plastic) 96-well plates will be used. This is because of 1) the number of cells/96-well is relatively low, and 2) fluorescence is scattered/"diffused" within the plastic, resulting in poor overall signal and loss of "experiments window", particularly if the differences in ROS production between your control and experimental treatment(s) are not that great to begin with. Cell culture plates with dark (black) walls and clear bottom (e.g. from Grainer Bio-One) gives much better readouts (at least in our hands).
Hope this will help you to optimize your experiments...Best of luck!
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