Quantitating secretion rates of individual cells: design of secretion assays.
To observe events occurring in the microenvironment surrounding individual cells, a mathematical framework has been developed describing the behavior of a compound following its secretion by a single cell. This description is based on the diffusional and binding processes taking place in the vicinity of the cell surface. It allows prediction of the rate of capture and accumulation of a secreted compound around a single cell. This concept provides the basis for the design of two experimental assays for measuring single-cell secretion rates: (1) Cells are immobilized in hydrogel microbeads which contain capture sites for the secreted compound; and (2) artificial receptors are bound directly to the cell surface which are capable of binding molecules secreted by individual cells. This general methodology is developed in the specific case of the model organism Saccharomyces cerevisiae secreting a heterologous protein, but can be applied to any cell/secreted protein combination. Binding studies have shown that approximately 2 x 10(5) of these artificial receptors can be attached to the surface of a single yeast cell. At this surface density of a putative artificial receptor, it is predicted that single-cell secretion rates of 47 molecules/cell/sec of a 150 kDa protein can be detected. Simulations indicate that a microbead loaded with 5 x 10(6) capture antibodies will result in detection of secretion of this protein at rates as low as 4 molecules/cell/sec.
Frykman
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Biological Process Technology Institute, 240 Gortner Laboratory, 1479 Gortner Avenue, St. Paul, Minnesota 55108, USA.
Srienc
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D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D006041:Goats; D006801:Humans; D007074:Immunoglobulin G; D051379:Mice; D008954:Models, Biological; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
D007074:Immunoglobulin G; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins