Method for optical measuring variations of cell membrane conductance

摘要

The instant invention refers to an optical method to extrapolate cell membrane conductance by indirect measurement of changes in transmembrane voltage, upon exposure of a cell sample to electric current pulses. The method is advantageously used for evaluating the activity of molecules able to alter, directly or indirectly, membrane permeability. A specific field of application is the screening of candidate compounds putatively acting on ion channel activity. In particular, it is open to the study of all ion channels with no limitations on the mechanisms of activation or to the ion species involved. The method is also advantageously used for evaluating a cell status, namely a differentiative or a pathologic status.

主权项

1. A method for measuring subcellular variations of the membrane conductance in a cell sample upon exposure to a compound, comprising the steps of:
a) labelling the plasma membrane of said cell sample with a voltage-sensitive dye, able to generate optically detectable signals indicative of local transmembrane potential with subcellular spatial distribution; b) exposing said cell sample to an electric current pulse with a constant current generator, wherein the total duration of the pulse is >20 milliseconds and its intensity is sufficient to evoke local transmembrane potential changes with no need to control their local intensity and spatial distribution; c) acquiring at least one image of the optically detectable signals generated by the voltage-sensitive dye, before step b) and at least one image of the optically detectable signals generated by the voltage-sensitive dye, during step b) by means of an array of photo-detectors; d) generating spatial maps of said local transmembrane potential changes of step b) by the optical images of step c); e) exposing said cell sample labeled in step a) to said compound; f) conducting said steps b), c), and d) on said cell sample exposed to said compound; and g) evaluating local changes in membrane conductance by comparing spatial maps obtained in step d) of said cell sample not exposed to said compound, with those obtained in step f) from said cell sample exposed to said compound.