Hi,

DCFDA is a non fluorescent lipophilic stain, crossing plasma membrane freely.

Once in the cytosol, DCFDA is hydrolyzed by esterases in DCF which is hydrophilic. It is then retained in the cytosol. DCF is oxidized by

cytosolic ROS and becomes fluorescent.

A difference in fluorescence intensity can mean two things,

1. Uptake and retention of DCFDA is lower in treated cells than in untreated cells. As penetration of DCFDA is a passive process, it means that cytosolic esterases responsible for the hydolysis of acetate groups of DCFDA are less active, leading to a lower accumulation of CDF in treated cells, then to a lower production of fuorescent compound.

2. Treated cells produce less ROS, then DCF is less oxydized to fluorescent compound.

As you stain for 30 min, we can guess the activity of esterases are equivalent, or compensated if there is an initial difference in activity, thus we can assume that treated cells produce fewer reactive oxygen species.

Cheers,

Oliv