9 investigators in physiology and immunology require a new cytofluorograph. The funded work of this group encompasses many topics in leukocyte biology and clinical medicine (arthritis, psoriasis, leprosy, leishmaniasis, lymphoma, vascular disease, diabetes, and transplantation). The cytofluorograph will be used in 3 types of experiments: 1) Characterization of primary cell populations by multiparameter analysis. Primary cell populations will be analyzed at the single cell level in terms of size and right angle scattering, cell cycle, and expression of two or more defined surface antigens. Such analyses provide quantitative and detailed descriptions of populations under study which include: neutrophils, macrophages, B lymphocytes, dendritic and endothelial cells, keratinocytes, Hodgkins' lymphomas, and different types of lytic or killer cells. Multiparameter analyses are required in the following physiological studies: a) binding and uptake of fluorescent ligands to phagocyte receptors, b) changes in intracellular calcium in macrophages and lymphocytes upon appropriate stimulation, c) stimulus-dependent alterations in specific cell surface antigens such as receptors for immune complexes, lymphocyte activation antigens, MHC products. 2) Production of monoclonal antibodies. The retrieval of demanding monoclonals is facilitated when such parameters as light scatter, cell cycle, and second surface antigens are used to identify cells that react with a hybridoma. Current goals are antibodies which a) identify dendritic cells and epidermal Langerhans cells, b) define functional epitopes on activated lymphocytes, c) distinguish endothelial cells, macrophages, and keratinocytes in different functional states. 3) Purification of defined populations by cell sorting. With many parameters to pinpoint a specific population or functional state, we would be able to sort well-defined subsets of cells. Subpopulations that require sorting include: B and T lymphocytes during their response to defined stimuli, dendritic cells in different states of maturation, Hodgkins' lymphoma, leukocytes in biopsies of infected tissue, macrophage parasitized with organisms labeled with vital fluorochromes. Sorted populations proteins tested for: a) functional in culture, b) expression of specific cytoplasmic proteins and mRNA's using antibody staining and in situ hybridization, and c) capacity to be cloned as specific antigen-responsive cells, or as cell line variants.
