Two new techniques for internalizing metallochromic indicators into the cytosol of mammalian cells are described. One method consists of hypertonically treating the cells in the presence of the
indicator, followed by a hypoosmotic treatment. The second method consists of incubating the cells at high density in a concentrated
indicator solution in physiological saline. Using either method,
arsenazo III or
antipyrylazo III was internalized into
Ehrlich Ascites tumor (EAT) cells at concentrations yielding measurable differential absorbance changes which correspond to changes in the intracellular Ca2+ concentration. In the case of
antipyrylazo III, the amount of
indicator internalized ranged between 140 and 350 microM, and was dependent on the metabolic state of the cell during loading. Control and loaded cells possessed virtually identical
ATP/
ADP ratios, as measured by high performance liquid chromatography (HPLC) in
cell extracts.
Antipyrylazo III was also internalized by rat hepatocytes without detectable cell damage. Treatment of metabolically active EAT cells with the
calcium ionophore A23187 results in only a slight increase in the intracellular free Ca2+ concentration, [Ca2+]i, whereas treatment with the
calcium ionophore ionomycin induces a substantial but transient increase in the [Ca2+]i. In contrast, metabolically inhibited EAT cells show a large rise in the [Ca2+]i upon addition of
A23187. Thus, these techniques offer another way of measuring intracellular free Ca2+ changes in mammalian cells and may prove useful, especially where concentrations of free cytosolic Ca2+ larger than 1 microM are expected.