Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.
Keywords: doublet discrimination; flow cytometry; light scatter; viable DNA dyes.
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