Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

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Abstract
Summary The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.