From: Howard Shapiro (hms@shapirolab.com)
Date: Wed Dec 24 2003 - 17:58:55 EST
YaeJean Kim wrote: >I have a question about granules from neutrophils or eosinophils. Does >the flow can detect the granules released from these cells?(maybe this is >the question regarding the lowest limit of size for particle detection by >flow) I have purified neutrophils and eosinophils run by flow and some tubes >activated with antigens I see little debries like group of particle with >decreased FSC. > >Could they be granules? IF so, can I somehow stain the granules and see >them? In the 1880's, Paul Ehrlich classified blood granulocytes based on their staining properties. The granules in acidophils (later commonly known as eosinophils) were noted to have a high affinity for acid dyes such as eosin. Eosin is actually a fluorescein dye with halogens on the ring, and, as Zbigniew Darzynkiewicz is likely to mention in response to YaeJean's posting, fluorescein stains eosinophils very nicely provided the cells are permeabilized, and should therefore stain free eosinophil granules. The component in eosinophils that attracts acid dyes is eosinophil basic protein, and, if you had a labeled antibody to that, you could stain the granules with it; however, because the affinity of the granules for fluorescein is so high, you might not need the specificity provided by an antibody. The granules in basophils have a high affinity for basic dyes, due to the granules' content of sulfated glycosaminoglycans; dyes such as the methylene azure dyes and toluidine blue are concentrated on or in basophil granules, with the result that spectral shifts (metachromasia) occur due to interactions between the pi orbital systems of dye molecules in close proximity. Ehrlich called neutrophils neutrophils because he believed that the acid and basic organic dye molecules in his stain mixtures formed complexes, producing what he called a "neutral stain". This is not entirely correct. The granules of neutrophils don't have particularly high affinity for either acid or basic dyes, and thus do not stain strongly with either type of dye. If you wanted to detect neutrophil granules specifically, you'd be best off using an antibody to some structural component that was either absent from, or present in only small amounts in, both basophil and eosinophil granules. The granules are probably too small to produce much of a forward scatter signal, although, since they are the principal contributors to the side scatter signal from granulocytes, you'd probably be able to pick them up by side scatter. For the eosinophil granules, a fluorescein signal should be strong enough for triggering. -Howard
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