pH dynamics in the lysosomes and cytoplasm of tissue culture cells measured intravitally using the indicator dye neutral red

A method of pH determination in the cytoplasm and individual lysosomes of intact living cultured cells is proposed using an indicator dye--neutral red (NR). The pH value of an investigated structure stained with NR is from the ratio between its optical densities at two wave lengths, the one corresponding to the isobestic point (lambda = 470 nm), and the other--to the absorption maximum of the neutral form of the dye (lambda = 450 nm). This method enabled us to make pH determination in the range from 4.5 to 7.2; the accuracy of the method reaches +/- 0.36 at pH 4.7 and diminishes to +/- 0.16 at pH much greater than 5.5. Using this method for the culture of pig kidney embryonal cells and Chinese hamster fibroblasts a heterogeneity of lysosomes was found with respect to the average pH value, oscillating ultradian changes in intralysosomal pH values, and the relationship between the changes and the size and average pH values of lysosomes. It is shown that the accumulation of NR in lysosomes depends on the pH gradient between lysosomes and the cytoplasm, which diminishes with the development of non specific cell response under the influence of some agents) high NR concentrations, inhibitor of glycolysis, or uncouplers of oxidative phosphorus lotion).