This proposal is to extend applicants investigations of a new bioanalytical methods: gel microdroplets (GMDs) and flow cytometry, which provides measurement of (1) clonal growth at the single cell level, and (2) secretion/release of molecule from single cells. The method is to provide population distributions of growth and secretion/release behavior and can isolate (sort) based on growth and/or secretion/release of molecules. The investigators claim that GMD-based measurements can significantly expand the applicability of flow cytometry, as GMD/flow cytometry is responsive to individual cells and their immediate environments (e.g. other cells, gel matrices with captured molecules). New information (subpopulations with different growth and secretion/release) is obtained in addition to conventional information (e.g. average growth rate), which also creates the possibility of replacing tedious methods (e.g. petri dishes, microtiter wells) with the GMD-based methods. Key attributes of GMDs are: (1) microscopic size with high permeability (therefore fast diffusional response); (2) physically manipulable (GMDs can be treated much like cells), allowing (a) use or ordinary laboratory procedures (e.g. pipetting, centrifugation), (b) incubation conditions ranging from in vitro to essentially in vivo, and (c) diverse possibilities for measurement (e.g. microscopy, flow cytometry); (3) suitability for adapting established flow cytometric fluorescence staining protocols; (4) suitability for subpopulation determinations, and therefore potential for directly making determinations on mixed cell populations. The investigators note that the GMD/flow cytometer method should find major applications in biomedical research, and has potential for clinical applications. Research possibilities include: (1) determination of subpopulations of secretors of specific proteins, and (2) isolating secreting cells (e.g. hybridomas producing antibodies). Clinical possibilities include: (1) antimicrobial susceptibility of individual """"""""primary"""""""" microorganisms taken from patients (particularly for """"""""slow growers"""""""" such as tuberculosis and yeast from immune-compromised patients), and (2) cancer tests (population distribution of chemosensitivity of cancer and normal cells, of cancer cell microcolonies).
