Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, 04510 Mexico, D. F., México.
J Bioenerg Biomembr. 2010 Oct;42(5):419-32. doi: 10.1007/s10863-010-9311-x. Epub 2010 Nov 10.
Different methods to estimate the plasma membrane potential difference (PMP) of yeast cells with fluorescent monitors were compared. The validity of the methods was tested by the fluorescence difference with or without glucose, and its decrease by the addition of 10 mM KCl. Low CaCl₂ concentrations avoid binding of the dye to the cell surface, and low CCCP concentrations avoid its accumulation by mitochondria. Lower concentrations of Ba²+ produce a similar effect as Ca²+, without producing the fluorescence changes derived from its transport. Fluorescence changes without considering binding of the dyes to the cells and accumulation by mitochondria are overshadowed by their distribution between this organelle and the cytoplasm. Other factors, such as yeast starvation, dye used, parameters of the fluorescence changes, as well as buffers and incubation times were analyzed. An additional approach to measure the actual or relative values of PMP, determining the accumulation of the dye, is presented.
比较了使用荧光监测器估算酵母细胞质膜电势差（PMP）的不同方法。通过有无葡萄糖的荧光差异以及加入 10 mM KCl 导致的降低来验证方法的有效性。低浓度的 CaCl₂可避免染料与细胞表面结合，低浓度的 CCCP 可避免其被线粒体积累。较低浓度的 Ba²+产生与 Ca²+类似的效果，而不会产生源自其转运的荧光变化。不考虑染料与细胞结合和线粒体积累的荧光变化会被它们在细胞器和细胞质之间的分布所掩盖。还分析了其他因素，如酵母饥饿、所用染料、荧光变化的参数以及缓冲液和孵育时间。提出了一种测量实际或相对 PMP 值的附加方法，该方法通过确定染料的积累来实现。
