I would like to see the increase in the ROS level in organotypic slice culture upon treatment with my compound. This compound is extremely precious, as it takes months and months produce in the lab and the quantity I get at the end will be very small.
I have to treat the slices for 72 hours with the compound and I would like to see the changes in the ROS level every 6 hours. My questions will be the following:
Is it possible to add H2-DCFDA (100µM) in the medium for 2 hours and then wash the slices and capture the 0 time point image? Then If I treat the same slices with my compound, is it possible to capture images without losing the intensity of the dye for 72 hours?
Not a good idea. This compound react with different radicals in different way and auto fluorescence increase with time. It produce itself radicals too as a part of the reaction mechanism. Please refer to Wardman FRBM for an extensive review about this molecule. Better monitoring GSH / free thiols levels- there are probes used for tracing stem cells which apparently are not toxic for them . Refer to the Molecular Probes catalog.
Good luck!
As suggested by Miriam, DCFDA shows cross-reactivity with many free radicals (ROS). You can use to determine till 48 hrs. The concentration that i keep is not more than 10uM. But the stimulus (ROS generator) generate very low ROS (low nutrients/serum and glucose free medium).
However the detection limit depend upon the stimulus use use for trigger.
For molecules like PMA/HOCl, we used different procedure. Since these are high ROS inducers. We treat the cells with the stimulus (PMA/HOCl) for various time points. after every 4 hrs we treat the cells with DCFDA and compare with parallel vehical control.
This may not be real time but, you can relatively determine ROS levels with respective control.
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