Flow cytometric assay of lung macrophage uptake of environmental particulates

We sought to establish a quantitative method using flow cytometry to study uptake of environmental particulates by alveolar macrophages (AMs). We used right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz, and diesel particulates. After incubation with TiO2 in vitro, AMs showed dose-dependent increases in both cell-associated particles visualized by microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4, 8, 16, 32, and 80 micrograms/ml, respectively, = 2 +/- 0.1, 4.0 +/- 0.5, 5.5 +/- 0.5, 9.1 +/- 2.5, 14.3 +/- 0.9; mean +/- SEM). Similar results were obtained with quartz and diesel particles. A strong correlation was observed between particle load per cell and AM RAS after uptake of fluorescent latex beads or fluorescent TiO2 (coated with BODIPY-BSA) (R2 = 0.984, 0.997, respectively). Using this technique, we found AM uptake of environmental particulates to be substantially greater than that of a panel of myelomonocytic and epithelial cell lines, consistent with their physiologic role in pulmonary defenses. RAS measurements have also identified both calcium-dependent and calcium-independent components in AM interactions with inert particles. Although this technique does not allow precise quantitation of particle number or mass per cell, flow cytometric analysis of relative increases in RAS is a useful tool to study AM interactions with a variety of environmental particulates.