ROS accumulation - ROS reactive oxygen species accumulation in cells (Mar/24/2010 )
Hello,
I am looking for a readout to assess the cumulative increase and/or damage done by Reactive oxygen species in the cell. Specifically, I would like to inject this compound into the living animal, then subject the organ of interest to oxidative stress, sacrifice the animal, fix the tissue and would like to be able to see the amount of ROS production or extent of ROS damage in the cell. All methods I saw so far only detect the ROS in the present state of the cell, but what I want is basically a recording of the history of ROS accumulation or damage in the cell over a time course of, let's say 1-2 weeks. I was thinking the DCF assay might be the way to go, but I read that DCF is bad for cells and they will not retain DCF in the cell, meaning they won't be a measure for the cumulative increase of ROS. I also thought about taking time points and to add the ROS measurements up for a final tally, but I am afraid that the ROS increase at a certain time point in the cell will be too small to detect. This is the main reason why I am interested in cumulative measurement.
If the above assay is impossible, what would be the best marker that I could use in imaging experiments to visualize cells that had increased ROS levels?
It would be great if someone could help!
Jangmugi
Evrogen has a vector for measurement of intracellular ROS (in the form of hydrogen peroxide) in different cellular compartments. I have never used it, but here is some information on it:
http://www.evrogen.com/products/HyPer/HyPer.shtml
It's also super expensive :\
Thanks Bill Nye,
the problem with the genetically encoded systems is that the cells I am interested in are difficult to transfect (auditory hair cells). Furthermore, HyPer still does not allow me to detect cumulative ROS damage or accumulation in the cells. But thanks for the reply: I have never heard of HyPer before and actually got some new ideas out of that.
I would appreciated other comments!
Jangmugi
And yeah, the HyPer system is expensive. It is just a vector, god dammit!
jangmugi on Mar 24 2010, 11:58 AM said:
I am looking for a readout to assess the cumulative increase and/or damage done by Reactive oxygen species in the cell. Specifically, I would like to inject this compound into the living animal, then subject the organ of interest to oxidative stress, sacrifice the animal, fix the tissue and would like to be able to see the amount of ROS production or extent of ROS damage in the cell. All methods I saw so far only detect the ROS in the present state of the cell, but what I want is basically a recording of the history of ROS accumulation or damage in the cell over a time course of, let's say 1-2 weeks. I was thinking the DCF assay might be the way to go, but I read that DCF is bad for cells and they will not retain DCF in the cell, meaning they won't be a measure for the cumulative increase of ROS. I also thought about taking time points and to add the ROS measurements up for a final tally, but I am afraid that the ROS increase at a certain time point in the cell will be too small to detect. This is the main reason why I am interested in cumulative measurement.
If the above assay is impossible, what would be the best marker that I could use in imaging experiments to visualize cells that had increased ROS levels?
It would be great if someone could help!
Jangmugi
I guess the problem you have is that something reactive in a surrounding of a lot of things to react with will not stay there for long.
DNA damage by ROS gets repaired, Proteins get degraded, Lipids are hard to properly quantify (and I assume they also get exchanged if oxidized..)
I would probably go with looking for oxidative modification of genomic DNA - but 1-2 weeks is a long time for any assay in that direction.
DCF is good in tissue culture, but it does not measure "previous" ROS (since those are gone).