>An investigator is looking at macrophages using FITC-conjugated (2 micron) >bead uptake plus ED-1 positivity as indicators of viable macrophages in a >sample that consists mostly of what morphologically appear to be macs. Some >of the ED-1 positive cells don't take up beads and he wants to be assured >that these ED-1 positive/bead uptake-negative cells are viable and not >binding the ED-1 (which is directly conjugated with phycoerythrin) >non-specifically as dead cells might. It has been suggested that he use >ethidium monoazide bromide (EMA) as an indicator of viability (the light >scatter properties of cells from some of the treated animals precludes using >scatter as a viability indicator). > >The plan is to first stain the cell samples with EMA, fix and permeabilize, >followed by staining with ED1. My question is, can we distinguish the PE >fluorescence from that of EMA? The Molecular Probes web site indicated the >fluorescence emission of EMA is around 625 but a reference they list >indicates that DNA-bound EMA has a fluorescence emission peak more like 600. >At 625 we might have a chance of distinguishing EMA from PE but I don't >think so if the EMA Em peak is 600. I haven't seen an emission spectrum for >EMA so can't tell how much PE will extend into it, or it into PE. > >Can this be done on a standard flow cytometer (we have a FACSVantage)? If >so, I suppose we would gate on the EMA negative cells first and then look at >the FITC/PE staining of those cells (?). > >I assume all three - FITC, PE, and EMA - can be excited at 488. > >Thanks for any advice. > >Ray Hester >rhester@jaguar1.usouthal.edu The answers to all your questions are yes, I believe. You can do this with the 488 laser on the Vantage. And you can distinguish EMA from PE--we do this routinely. Of course, there will be substantial compensation, but with a reasonable PE signal you can see it fine. -- Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 203-688-2748 (fax) mark.shlomchik@yale.edu
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