Chris Hathaway asks: "....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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