Anchored Cell Analysis and Sorting Cytometer

Kavanagh, Terrance

Abstract

The purpose of this application is to receive monetary support for the purchase of an anchored cell analysis and sorting interactive laser cytometer. There are many research projects within this institution that can benefit from shared access to such an instrument. The users of this instrument will take advantage of its unique interactive capabilities which allow for the sorting and physiological characterization of adherent tissue culture cells in situ, under conditions which best represent their normal physiology. This is accomplished by this instrument through a computer-controlled laser connected to an inverted fluorescence microscope with a heated stage whose movements are also computer-controlled. The laser on the instrument will be used to sort adherent cells in situ via laser ablation of unwanted cells; to photobleach carboxyfluorescein labelled cells in order to study intercellular communication; to photobleach fluorochromes in cell membranes as a measure of lateral diffusion and membrane fluidity; and to photolytically activate so-called caged compounds to release substances within the cell to study their effects on cell physiology and behavior. The instrument will also be used to measure various physiological parameters in adherent cells in real-time such as changes in intracellular calcium and pH after mitogen stimulation; changes in intracellular calcium, glutathione, and peroxide after exposure to toxic substances or reactive oxygen species; and enzyme kinetic studies such as glutathione S-transferase activity. While conventional flow cell sorting facilities are available at this institution, measurements on adherent cells that require the cells be anchored to a substratum in order to display normal physiology or that require specific cell-cell interaction are either impractical or not possible with flow-based instruments. The anchored cell analysis and sorting cytometer will complement the conventional flow cell sorters already available, by providing for evaluation and sorting of adherent cells in situ.