Fig. 2. Flow cytometric analysis and sorting of low and high M540 fluorescent viable sperm cell subpopulations. Sperm cells were incubated, analyzed and sorted using a FACS Vantage SE as described in Materials and Methods. Sperm-specific events with fluorescent properties in M540 fluorescence (membrane fluidity) and Yo-Pro 1 fluorescence (viability) were continuously recorded and sperm cells in specified regions (gray circles) were sorted and collected at room temperature (a). Within 10 minutes, the sorted and collected sperm cells were re-analyzed for low M540 fluorescence (b) as well as for high M540 fluorescence (c) demonstrating that cells remained viable and did not alter M540 fluorescent properties. The sorting efficiency was >99% for the low M540 and >95% for the high M540 fluorescent sperm subpopulations, respectively. Under a confocal microscope the unsorted sperm cells contained low and high M540 fluorescent cells (d), whereas the subpopulation sorted for low M540 fluorescence showed very dim fluorescence (e), and the subpopulation sorted for high M540 fluorescence showed bright fluorescence (f).