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"....would like to evaluate circulating neutrophils, specifically
quantify them according to their activation state. Is there any way
to determine the number (percentage) of cells that are primed,
activated, etc. from a blood sample? "
Chris, this is a really easy thing to do in flow cytometry - there
are several probes that are excellent for the job, namely
dihydrorhodamine 123, hydroethidine and dichlorofluorescin diacetate.
These probes allow you to measure the respiratory burst of
neutrophils. The methods are very straight forward. You incubate hte
cells with the probe, stimulate the cells with an appropriate
stimulant (PMA, fmlp, etc) and measure the change in fluorescence
after a few minutes. The resting cells will exhibit a fluorescence
level equal to the basal respiration of the cells. If your cells are
activated, then this "resting" fluorescence will be high. We observed
this some years ago when measuring hte difference between "resting"
ie unstimulated neutrophils taken from gum diseased periodontal sites
as opposed to "clean" sites. (Infect.Immun.56:156-160 (1988))
Essentially you can measure several reactive oxygen or reactive
nitrogen molecules using fluorogenic probes. Detailed methods can be
found in the Handbook of Flow Cytometry Methods (Wiley Liss), or
Methods in Cell Biology, 41:437-447, 1994.
If you have specific questions give me a call.
Regards
Paul Robinson
J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson@flowcyt.cyto.purdue.edu PH:317-494 6449 FAX:317-494 0517
web http://www.cyto.purdue.edu
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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