Hello,
I am using amplex red assay to measure H2O2 in extracellular medium.
The cells (HepG2) were treated with high glucose and high insulin over night to achieve insulin resistance (checked with western blot, and glucose concentration measurements). As controls I have normal cells (grown in low glucose), or cells grown in high glucose only without insulin.
The cells that have been treated with high glucose only give a nice increase in ROS by about 30-40%. However, cells treated with insulin and high glucose ROS levels are the same or less than low glucose control. It seems as if my signal is being quenched. Maybe too much ROS is being generated? What do you suggest?
Dear Sara,
first of all. your choice of Amplex Red for measuring extracellular H2O2 is very good. As far as I read your data, induces high glucose an increase in extracellular H2O2 and additonal treatment with insuling reduces or inhibits this increase. These findings seem tempting and good to me. Nothing to worry. I do not think that you increase your ROS production with additional insulin treatment to a point where too much ROS are produced, so your probe will get used up.
If you have any further questions, feel free to ask any time.
All the best and stay healthy,
Marc
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