Measurement of changes in cytoplasmic free Ca²⁺ in fused cell hybrids

Abstract

The investigation of many problems in cell biology requires a method for studying chemical ‘events’ in living cells. For example, the measurement of cytosolic free Ca²⁺, using Ca²⁺ indicators in giant cells, has provided vital evidence for the role of this ion in the activation of cells by electrical and chemical stimuli¹. However, the incorporation of Ca²⁺ indicators into small cells (diameter <20 µm), without causing irreversible damage, has proved extremely difficult¹. Recently, an elegant new technique has been reported² for entrapping within small cells a fluorescent Ca²⁺ indicator after hydrolysis within the cell of a membrane-permeant ester of the compound. We have developed a method, using Sendai virus, for fusing cells with erythrocyte ghosts containing photoprotein Ca²⁺ indicators and other membrane-impermeant chemiluminescent-labelled indicators^3,4. Because of the sensitivity of chemiluminescent analysis, these indicators can be used at concentrations which do not significantly disturb the chemistry of the cells. The ultimate aim was to establish a cell system in which free Ca²⁺, oxygen radicals and cyclic nucleotides could be directly monitored. Here we report the incorporation of the photoprotein, obelin⁵, an indicator of free Ca²⁺, into small mammalian cell hybrids.