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As you know, glutaraldehyde induces significant cellular
autofluorescence. My question: Is there a known flow cytometry
investigation which successfully demonstrated a method to eliminate
the autofluorescence associated with glutaraldehyde-fixed RBCs? Or,
blood cells of any kind? Or, cells of any kind?
Glutaraldehyde, I've experiences, does not inhibit specific binding
of FITC-annexin V to membrane surface-exposed phosphatidylserine
(PS). In a previous pilot study, glutaraldehyde-fixed echinocytes
(abnormal RBCs induced by ATP-depletion of RBCs)(characterized by PS
translocated to the outer membrane leaflet) were highly fluorescent;
and, fresh, unfixed normal RBCs lacked fluorescence. This suggests
that glutaraldehyde induced significant autofluorescence in both the
_normal_ and the _echinocytic_ (abnormal) populations of GA-fixed
RBCs.
Glutaraldehyde, the accepted fixative for RBCs, does _not_ cause
transport of small proteins (like annexin V) intracellularly for
access to PS in the inner membrane leaflet as does formaldehyde or
paraformaldehyde. I will need to use glutaraldehyde in the future.
Accordingly, I'm hoping you can advise me of a _chemical method to
eliminate autofluorescence associated with glutaraldehyde-fixed
RBCs_, or, alternatively, appropriate fluorochromes with wavelengths
beyond that of the glutaraldehyde spectrum, or, an instrumentation
technique.
My current affiliation is with the Uniformed Services University of
the Health Sciences, Bethesda, MD.
Thanks so much.
John H. Boucher, DVM, PhD
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Steve Kelley kelley@flowcyt.cyto.purdue.edu
Purdue University Cytometry Laboratories (317) 494-0757 -- voice
B050 Hansen LSRB, Purdue University (317) 494-0517 -- fax
West Lafayette, Indiana, 47907
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