Flow lysometry and its biomedical applications

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DESCRIPTION (provided by applicant): Single-cell analysis by flowcytometry has revolutionized the measurement of complex cell populations. Only limited probes are available for the measurement of cytosolic components in single cells. While cellular components such as ATP can be measured sensitively in a suspension of cells with permeabilized plasma membranes, the determination of the distribution of such compounds in individual cells in a heterogeneous population is not available. We propose the development a novel technique, flow-lysometry, by a multi- disciplinary approach, combining cell biology and nanotechnology developed by micromechanical engineering, to measure cytosolic components of single cells. Our preliminary data show that we can establish lysis of individual cells in a flow channel and detect ATP by a chemiluminescent pulse similar to an event in flowcytometry. The synchronization of conventional flowcytometry with flow-lysometry will allow measurement of surface components in conjunction with cytosolic components in a heterogeneous cell population. As specific aims we propose to 1. develop a device for flow-lysometry, 2. synchronize flow-lysometry with flowcytometry, and 3. test flow-lysometry on a heterogeneous cell population. Fabrication of dedicated micro fluidic channel flow-lysometry chips interfaced with a conventional flowcytometer will allow synchronized cell- detection, cell-lysis, and cytosolic component sensing. We will test the efficacy of this technology by measuring ATP levels in phosphatidylserine (PS) exposing cells in the heterogeneous population of sickle red blood cells. Our research will establish this novel technology to measure cytosolic components in defined, (subpopulations of) individual cells. We envision that this technique can be applied to various types of cells measuring many different cytosolic components.