Labeling rat alveolar macrophages with fluorescent microspheres

Rats were exposed for various periods (5, 20, 60, and 120 min) to an aerosol of fluorescent polystyrene microspheres (FPM) with nominal diameter 1.07 μm. All the animals were killed 40 h later and the lungs were lavaged 10 times with physiological saline. Washings 1-2 and 3-10 were combined. The numbers of alveolar macrophages (AM) recovered by lavage were measured with a Coulter counter. Cytospin slides of cells in the lavage fluid were prepared and used to determine the fraction of recovered AM that contained FPM (labeling index, LI) and the FPM/AM profile. The same parameters were also measured by flow cytometry. There was good agreement between determinations of LI and FPM/AM by manual scoring methods and by flow cytometry. Samples of lavage fluid were digested with sodium hypochlorite (bleach), and aliquots of the resulting digests were filtered. Examination of the filters with epifluorescence microscopy enabled the total numbers of FPM present in the various lung washes to be determined. The lavaged lungs were also digested with bleach, and the number of FPM remaining was estimated by manual scoring only. Flow cytometry could not be used for this purpose, due to the presence of cellular debris. After exposure to airborne FPM at the concentration used for 2 h, less than half the recovered AM contained FPM. For studies of AM kinetics it will be necessary to achieve higher LI values and numbers of FPM per cell than were obtained in the present study.