The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells

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Abstract
Peripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90° light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90° scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90° scatter than forward scatter. Simultaneous use of forward and 90° scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90° scatter alone, forward scatter alone, and forward and 90° scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90° scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.