Isolation of Clara cells from rat lung using flow cytometry.

Clara cells are involved in the cytochrome P-450-dependent metabolism of certain lung toxins, e.g. ipomeanol [I], but studies have mainly been on their action on whole lung tissue. Attempts to isolate Clara and other lung cells have in the past produced only partial enrichment owing to the limitations of purification by density gradients and centrifugal elutriation [2, 31, although, more recently, mouse Clara cells of high purity have been isolated by differential attachment to IgG-coated dishes [4]. Fluorescence-activated flow cytometry has been used to separate fibrosarcoma cells based on their glutathione content [ 51 and hepatocytes based on their mixed-function oxidase activities [6]. Lung type I1 cells have also been isolated after staining of their lamellar bodies with Acridine Orange [7] or phosphine 3R [8]. Based on the reaction of their glutathione content with monochlorobimane to a fluorescent product [9], subpopulations of Clara cells have now been obtained from rat lung by the use of fluorescence-activated flow cytometry. Single cell suspensions were obtained from the lungs of male Fischer F-344/N (200 g) rats after initial clearance of the lung vasculature by perfusion with Hanks' balanced salt solution and of macrophages by alveolar lavage through the trachea. A recirculating enzyme perfusion was performed via the trachea using subtilisin [lo] (0.33 mg/ml) for 30 min at 37"C, resulting in yields of 8 x lo7 cells/lung with a viability of 86% * 3% ( m e a n f s . ~ . , n = 11) based on Trypan Blue exclusion. Cells were reacted with monochlorobimane (10 p ~ ) for 2 min at 37°C and analysed on an Ortho Cytofluorograf 50H fluorescence-activated cell sorter using a krypton ion laser with an excitation wavelength of 407 nm and an emission long-pass filter with 50%T at 430 nm. Fig. 1A shows the 90" versus narrow-angle forward scatter for the unsorted lung cell preparation. A computer-defined region [ 11 was selected; the fluorescence characteristics of the cells from this region are shown in Fig. 1B. From this subset, the most fluorescent cells were sorted at a rate of about 15 cells/s from the marked region (-). Clara cells from this region were of >90% viability as judged by Trypan Blue exclusion and counting with the aid of a haemocytometer. Preparations remained viable in culture in supplemented Williams E medium in fibronectin-coated tissue culture chamber slides for 36 h. At the light microscope level, such cells were characterized by their Nitroblue Tetrazolium reductase activities after fixation of 30 s in acetone at 4°C and by their lack of staining with a modified Papanicolaou reagent. This visualizes lamellar bodies of the type 11 cells which were the major contaminating species. For electron microscopy, fixation was carried out in 2% (v/v) glutaraldehyde/osmium tetroxide followed by staining in uranyl acetate. Clara cells were identified by the presence of extensive smooth endoplasmic reticulum and secretory granules. By these criteria, such cells had a purity of > 90% and represented about 0.8% of the total. Lung perfusions with enzymes such as protease XIV and elastase gave poorer yields under our sorting conditions. Cells isolated in this way may be of use in the study of mechanisms of action of lung toxins, particularly those which require metabolic activation by the cytochrome P-450-dependent mono-oxygenases.