Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another result in self‐quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity of antibodies relative to one another on a highly labeled cell surface. Since self‐quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase‐sensitive detection method to investigate the impact of self‐quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti‐mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self‐quenching effects expected at high densities of FITC molecules. (This article is a U.S. Government work and, as such, is in the public domain n the United States of America.) © 1996 Wiley‐Liss, Inc.

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In patients with severe sepsis, big-ET plasma levels are markedly increased, even above those of multiple trauma patients, in close relationship to IL-6 and IL-8, and with significant correlation to renal function and pulmonary vascular tone.

Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals. In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)

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