Daniel H. Zimmerman

A simple procedure for labeling the lipid a moiety of lipopolysaccharide with 125I

A procedure for the iodination of lipid A is described. The iodinated product binds to rabbit spleen cells but not to red blood cells, and the binding to such spleen cells is inhibited by the presence of an excess of unlabeled lipid A. In dilute buffer solution, 125I-labeled lipid A appears to be aggregated because it migrates on Sephadex G-100 in the region of the void volume, whereas it migrates as would a molecule even smaller than cytochrome c upon electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. Evidence is presented that is consistent with the view that the iodine label is associated with the O-acyl groups of lipid A.

125I-lipid A binding by individual macrophage cells: Cell purification, culture, and autoradiographic analysis on a single surface

Abstract A method for studying the binding of 125 I-lipid A by individual macrophage cells is described. Cell populations adherent to glass coverslips were derived from the spleen and peritoneal cavity of mice. Macrophage cells within these populations were identified by their ability to ingest antibody-coated sheep red blood cells. Cell monolayers were subsequently incubated with 125 I-lipid A and processed for autoradiography. Analysis of the number, density, and distribution of silver grains indicated that macrophage cells were not equivalent in their ability to bind lipid A. While only 56% of the macrophage cells obtained from spleen exhibited lipid A binding, at least 93% of the peritoneal macrophage cells specifically bound lipid A. This simple method of isolation, identification, and autoradiographic analysis is readily amenable to other investigations in which the interactions between radiolabeled compounds and individual macrophage cells are of specific interest.