A non-disruptive technique for loading calcium buffers and indicators into cells

Abstract

Present methods for measuring or buffering intracellular free calcium concentrations are almost entirely limited to robust and well anchored cells which can tolerate insertion of ion-selective microelectrodes or microinjection of calcium indicators or buffers into one cell at a time. A very few types of small cells can be loaded with buffers or indicators during controlled lysis, but such procedures grossly perturb membrane integrity and soluble cytoplasmic constituents. Liposome fusion releases only trace quantities of the trapped solute into the cytoplasm and incorporates foreign lipid into the target cell membranes. I now describe a simple technique which loads Ca2+-selective chelators into the cytoplasm of intact cells in suspension and avoids the disadvantages of previous methods. The chelators are made temporarily membrane permeable by masking their four carboxylates with special esterifying groups which then hydrolyse inside the cells, regenerating and trapping the original chelators. The method is demonstrated on red cells, mast cells and lymphocytes.