Phagocytosis: Flow Cytometric Quantitation with Fluorescent Microspheres

Abstract

The phagocytosis of uniform fluorescent latex particles by pulmonary macrophages in the rat was analyzed by flow cytometric methods. The percentage of phagocytic macrophages and the number of particles per cell were determined from cell-size and fluorescence histograms. A comparison of in vivo and in vitro phagocytosis data showed that the percentage of phagocytic lavaged macrophages reflected the availability of instilled particles. With sodium azide used to model phagocytosis inhibition, it was shown that the percentage of phagocytic cells and the number of particles per cell can be determined simultaneously.

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

“…Confocal microscopy has shown that internal membranes within the cell fuse with plasma membrane during the course of particle ingestion, and that recycling endosomes are the primary source of membrane for enlargement of phagocytic cup [22]. Flow cytometric assessment of particle engulfment has to some extent replaced microscopic observation [23] particularly as the kinetics of uptake of fluorescent targets by phagocytes can be followed by instruments capable of time-resolved measurements in a stirred cuvette at 37°C [24]. These assays of phagocytosis by flow cytometry usually include measurements of fluorescence particle uptake by cells pre-incubated with cytochalasin D (CytD), an inhibitor of F-actin polymerization and phagocytic cup formation which allows the assay to distinguish engulfment of particles from adhesion.…”

Egr-1 abrogates the E2F-1 block in terminal myeloid differentiation and suppresses leukemia

“…Latex beads are visualized with the FITC filter, F-actin with the Texas Red filter, and nuclei with the DAPI filter. To quantify phagocytic cells, cells were subjected to flow cytometry (BD FACSCalibur) using Cell Quest Pro 5.2 software (Becton Dickinson, San Jose, CA, USA; Steinkamp et al, 1982).…”

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

“…For this reason, phagocytic function has been the object of intensive investigation, including the use of flow cytometry for studying phagocytic function associated with disease (1). Numerous flow cytometric techniques have been used to assess the ability of phagocytes to ingest various kinds of nonopsonized and opsonized particles, including fluorescent latex microspheres (2)(3)(4)(5)(6)(7)(8), bacteria (6,9 -13), yeast (14), fungi (15,16), sheep erythrocytes (17), apoptotic lymphocytes (18), and environmental particulates (19). Flow cytometric assays offer two major advantages over microscopy-based techniques in that (a) thousands of cells are examined in minutes and (b) other cellular features and functions can be concurrently assessed.…”