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I did some preliminary work on this with Don Ewert here at Wistar. We
examined the green fluorescence of a chicken B cell line stained with
rhodamine 123, using the FITC filters on a Coulter XL. Serum starvation
and/or treatment with the Na+/H+ antiporter inhibitor amiloride causes a
DECREASE in the green fluorescence of these cells within a short time,
say 6 hours or so. This decrease in Rh123 fluorescence is corroborated
with other flow cytometric indicators of apoptosis.
The change in fluorescence is remarkable when observed by fluorescent
microscopy. In the non-apoptotic cells, the Rh123 is compartmentalized
in the mitochondria, since Rh123 is a lipophilic cation, and the
interior of an intact mitochondrion is negative. The treated cells, on
the other hand, have dim fluorescence throughout the cytoplasm. It
appears that once the mitochondria have lost their ability to
concentrate Rh123, the dye is free to cross the plasma membrane along
the concentration gradient. This is enhanced by washing the cells with
PBS right before running them on the cytometer.
Like I said, it was preliminary work, but it did seem to work.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web