Simultaneous membrane capacitance measurements and TIRF microscopy to study granule trafficking at immune synapses

Abstract

Whole-cell capacitance measurements allow the direct measurement of exocytosis with high temporal resolution. An added benefit of the whole-cell configuration is the possibility to control the cytosolic free calcium concentration allowing examination of the role of intracellular calcium in a variety of processes. We have coupled this method with imaging of cytotoxic granule release using total internal reflection fluorescence microscopy (TIRFM) to identify the capacitance steps associated with cytotoxic granule release identified by TIRFM. This requires the use of fluorescent granule markers to identify cytotoxic granules and allows characterization of cytotoxic granule fusion and of the behavior of cytotoxic granules at the immune synapse prior to fusion. Combination of these methods enables the study of a number of processes relevant to the function of the immune synapse.