I am forwarding this message to the flow community from a user of our core
flow cytometry lab. Thanks in advance!!!
Michele A. Lutz
VA Medical Center
San Diego, CA
I need to measure pH (relative) of neutrophils endosomes/lysosomes
under various culture conditions. Since in some conditions, the lysosomes
may be neutral instead of acidic, I am concerned that dyes like the
"LysoTracker" of molecular probes will not load into the organelles very
well. Would I be better off using something like NERF-dextran or
SNAFL-dextran and hoping the neutrophils eat enough of the particles to give
me a reliable signal?
Do you have any references that will help me design the right loading protocol?
I think that the best choice may be the DM-NERF dextran conjugate
(70,000MW or 10,000 MW ?), which can be ratiometrically analyzed with dual
excitation (488 and 514) and single emission (570). Since it is
particulate, the meutrophils will have to eat it, which will mean that I
will mostly image secondary granules, which is just fine by me.
Any thoughts?
Thanks,
Robbie
Roberta A. Gottlieb, M.D.
Associate Member
Division of Biochemistry
Molecular & Experimental Medicine
The Scripps Research Institute NX7
10550 North Torrey Pines Road
La Jolla, California 92037
Tel. (619) 784-7929
Fax (619) 784-7981
website: http://www.scripps.edu/robbieg/
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