I actually compared and sorted EOS on the polarised / depolarised scatter from a HENE and a 488 air-cooled successfully in the EPICS Elite which also agreed with the autofluorescence in either 525 or 575 nm. The change in side scatter (and fluorescence) becomes actually much more pronounced after a fixative lyse such as BD Facslyse. If you look at log/log scatter plots (which one should do all the time) they cluster well apart from the neutrophils on the EPICS XL. We have actually stored all our whole blood data ungated. With the EOS usually being detectable as scatter cluster we could look at their CD4 distribution which we usually measured in PE-Cy5 if I remember correctly. I will have a look if I get hold of some of the old data. Gerhard -----Original Message----- From: Howard Shapiro [mailto:hms@shapirolab.com] Sent: 08 October 2002 00:05 To: cyto-inbox Subject: Re: A query? Lakshna Mahajan wrote- > can you please suggest that cells like eosinophils which have > autoflouresence be sorted based on this property itself using > flowcytometry? There are only two reports on that and i donot know the > reason for separation using this technique. is it because it becomes a > costly affair? Your reply will be of help to me. The original reports on identifying eosinophils based on high autofluorescence date back to the days when fluorescence filters were not very good and when side scatter measurements were not routine. Both of these problems have been corrected in modern cell sorters. In my experience, when using 488 nm laser excitation, it is possible to define an eosinophil population in unstained samples by gating on a display of side scatter vs. green (same filter as used for fluorescein) fluorescence; you could also use yellow (same filter as used for phycoerythrin fluorescence) instead of green. The green fluorescence is roughly the same order of magnitude as what would be expected from cells bearing a few thousand molecules of fluorescein, so you might want to try some low intensity fluorescein-labeled beads to make sure your instrument can detect signals in this range. The "Quantum" beads originally developed by Flow Cytometry Standards Corporation, and now made by Bangs Laboratories, are suitable for this purpose. Once you have a cell sorter, sorting eosinophils can be less costly than sorting many other cell types because no antibody reagents are needed, but the costs of antibodies are, after all, rather low when compared to the cost of the sorter. In a response to a previous inquiry about identification and sorting of eosinophils, I mentioned that they show weak expression of CD4, but did not mention that some people seem to think that CD4 expression occurs only on activated eosinophils, while others think CD4 can be detected on all eosinophils, but that levels of expression vary with activation state. As far as I know, eosinophils from mammalian and avian species show higher depolarized side scatter signals than do other cells, and can be identified and sorted if one adapts an instrument to measure both polarized and depolarized side scatter. The autofluorescence and CD4 issues could be resolved fairly efficiently by setting up an instrument to measure polarized and depolarized side scatter, green autofluorescence excited at 488 nm, and CD4, using either a PE-Cy5-labeled antibody with 488 nm excitation or an APC-labeled antibody with red (633/635 nm) excitation. If I can't get anybody else interested in playing this game, I'll try it myself after the overdue 4th Edition of Practical Flow Cytometry appears. -Howard
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