I have measured ROS levels of Chlorella using DCFH before and after exposed to heat stress with following intervals 3h, 6h, 9h, 24h and 48h. The level of ROS before the treatment was higher. How is that possible?
DCFH, or rather it's breakdown product has been shown to be somewhat artifact prone Article The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen...
I'm not familiar with Chorella, but depending on when the dye is being added, it may be breaking down. Alternatively, heat stress may be affecting intracellular iron levels. I might suggest trying a few different ROS indicators (dihydroethidium or Amplex red/HRP for extracellular ROS) to determine if what you're observing is actually ROS or just an artifact. Alternatively, looking at GSH:GSSG might provide with you more information about the redox status of the cells.
The ROS production before treatment is maybe due to Chlorella cell culture conditions such as medium osmoarity, shear stress, nutrient deficiency, etc..
@mohamad Issawi, but after 3 hours incubation the ROS level declined significantly to more than half.
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