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
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