Eighteen holders of NIH grants pursuing 20 NIH-funded projects (together with 9 others) are requesting funds to purchase an Anchored Cell Analysis and Sorting System (ACAS 570). The proposed instrument is the equivalent of a flow cytometer that works on cells growing on plastic or glass surfaces. Flow cytometers, working on suspended cells, necessitate the detachment of normally adherent cells from their growth substrate which clearly destroys the integrity and/or the distribution of surface antigens together with intercellular communication. This makes it impossible to sort a given cell type using surface antigen probes. The ACAS system consists of a fluorescence microscope with laser optics and associated computer control facilities. It scans the field of cells and measures their fluorescence parameters. From these data, it can be programmed to destroy cells that do not meet the required criteria. In addition, the apparatus can analyze fluorescence intensity distribution of a cell population, measure diffusion coefficients of cell surface antigens using fluorescence redistribution after photobleaching and use fluorescent dyes to measure intracellular components. Indeed, the versatility of the machine is only limited by the availability of fluorescent probes and the availability of the appropriate software for analysis. The ACAS 570 has a confocal microscopy option which will allow us to detect and analyze fluorescent probes in highly polarized cells and in cell with high background fluorescence. We propose a variety of investigations including: (a) the sorting of primary cell cultures to obtain homogenous cell cultures, (b) sorting of cells transfected with foreign genes from those not expressing those genes, (c) pH measurement of components of the endocytotic pathway through which surface-bound ligands pass, (d) calcium measurements after stimulation of cells with hormones and during cellular differentiation, (e) C-kinase investigations and (f) the measurement of the effects of cytoskeletal components on surface antigen migration. We also propose to use confocal fluorescence microscopy to extend current studies on (a) the distribution and targeting of surface antigens to various surfaces of epithelial cells, (b) the organization of the extracellular matrix, (c) the sites of degradation of blood-borne proteins by hepatocytes and (d) the organization of the cytoskeleton.
