Hammond T G, Majewski R R, Morré D J, Schell K, Morrissey L W
Department of Medicine, William S. Middleton Memorial V. A. Hospital, West Lafayette, Indiana.
Cytometry. 1993;14(4):411-20. doi: 10.1002/cyto.990140410.
The technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification. To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H(+)-ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction. Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations.
脉冲宽度分析技术是为优化细胞周期动力学中的细胞大小分辨率而开发的，此前尚未应用于诸如内体等小颗粒。偏移用于从正向散射脉冲宽度信号中减去一部分光束直径，以优化小颗粒的可视化和辨别。我们通过光散射参数的偏移脉冲宽度识别多个内体群体。具体而言，线性正向散射脉冲宽度测量显示，在大鼠肾皮质、大鼠肾乳头和蟾蜍膀胱腔内皮中至少存在两种内体群体。对数放大的正向散射脉冲宽度测量显示了这些信号的完整动态范围，分辨出了线性放大未显示的其他群体。为了确认观察到的内体与光学和电子噪声区分开来，我们检查了其生理功能。向内体提供ATP后，内体中的固有膜H(+)-ATP酶会使内体酸化。此外，对蟾蜍膀胱分选的内体群体进行电子显微镜检查，证实了该部分的同一性和同质性。通过多参数技术对内体群体进行流式细胞术分析，包括对结构参数的脉冲宽度分析和对包埋荧光团荧光的脉冲高度分析，可对多个内体群体进行识别、分离和定量。
