Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into...

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.