LABELING RAT ALVEOLAR MACROPHAGES WITH FLUORESCENT MICROSPHERES

Rats were exposed for various periods 15, 20, 60, and 120 mini to an a erosol of fluorescent polystyrene microspheres (FPM) with nominal diam eter 1.07 mu m. All the animals were killed 40 h later and the lungs w ere lavaged 10 times with physiological saline. Washings 1-2 and 3-10 were combined. The numbers of alveolar macrophages (AM) recovered by l avage were measured with a Coulter counter. Cytospin slides of cells i n the lavage fluid were prepared and used to determine the fraction of recovered AM that contained FPM (labeling index, LI) and the FPM/AM p rofile. The same parameters were also measured by flow cytometry. Ther e was good agreement between determinations of Il and FPM/AM by manual scoring methods and by flow cytometry. Samples of lavage fluid were d igested with sodium hypochlorite (bleach), and aliquots of the resulti ng digests were filtered. Examination of the filters with epifluoresce nce microscopy enabled the total numbers of FPM present in the various lung washes to be determined. The lavaged lungs were also digested wi th bleach, and the number of FPM remaining was estimated by manual sco ring only. Flow cytometry could not be used for this purpose, due to t he 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 II values and numbers of FPM per cell than were obtained in the prese nt study.