I have been trying to detect reactive oxygen species (ROS) in sperm using
a fluorescent dye.  I have used a derivative from 2'-7',dichlorodihydrofluo
rescein diacetate (H2DCFDA), the 5-chloromethyl H2DCFDA (CM-H2DCFDA, Molecular
Probes).  This dye should allow observation of ROS generation over time, since,
once oxidized, it is retained for a longer time in the cells as compared to
H2DCFDA and its others derivatives.
The sperm cells labeled with CM-H2DCFDA showed very low fluorescence intensity,
even when exposed to 93 micromolar of the dye.  this fluorescence intensity
was not increased in the presence of H2O2.  However, large increase in
fluorescence intensity was observed when the cells where incubated with high
amounts (8 % v/v) of DMSO at the loading time or prior to that.  I think the
fluorescence intensity increased due to the permeabilization of the cells and
not due to better solvation of the dye, since the same effect was not
observed by increasing pluronic acid added to the dye stock.
This particular derivative of H2DCFDA is relatively new and I have not found
in the literature any work describing itts use.  therefore, I would like to
know: Have you used CM-H2DCFDA? Have you used H2DCFDA or any other of its
derivatives? How do they compare?
Thank you,
Coralia Medeiros
Animal
Animal Sciences Department
University of Wisconsin
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