Phagocytosis and ATP levels in alveolar macrophages during acute hypoxia

SK Leeper-Woodford, JW Mills - Am J Respir Cell Mol Biol, 1992 - atsjournals.org
SK Leeper-Woodford, JW Mills
Am J Respir Cell Mol Biol, 1992atsjournals.org
Materials and Methods Cell Isolation Procedures PAM were obtained by lung lavage from
New Zealand white rabbits (2 to 5 kg) according to the method of Myrvik and associates (20).
In each rabbit, the lung washings removed by the lavageprocedure were centrifuged (400
xg, 10min), pooled, washed, and resuspended in phosphate-buffered saline (PBS).
Macrophages in each population were identified by cell size and shape, and viability of the
cells was determined by their ability to exclude trypan blue dye (21, 22). Purification and …
Materials and Methods
Cell Isolation Procedures PAM were obtained by lung lavage from New Zealand white rabbits (2 to 5 kg) according to the method of Myrvik and associates (20). In each rabbit, the lung washings removed by the lavageprocedure were centrifuged (400 xg, 10min), pooled, washed, and resuspended in phosphate-buffered saline (PBS). Macrophages in each population were identified by cell size and shape, and viability of the cells was determined by their ability to exclude trypan blue dye (21, 22). Purification and cultivation of the alveolar macrophages was done according to Edelson and Cohn (22) by suspending the cells in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (GIBCO, Grand Island, NY) and placing the suspension (1 x 106 cells/dish) on glass cover slips in 35-mm plastic culture dishes (Corning Glass Works, Corning, NY). Cells were incubated (95% air/5% CO2, 37 C) for 16 to 20 h to allow adherence of the alveolar macrophages in the culture dishes. Nonadherent cells and DMEM were then decanted, and the adherent cells were rinsed twice with warm (37 C, pH 7.4) Krebs Ringer bicarbonate (KRB). The experiments that followed the above procedures were completed within 24 h of PAM isolation from the rabbits.
The nonspecific esterase method according to Koski and co-workers (23) and the acid phosphatase enzyme staining technique using the Sigma Diagnostic Kit for cytologic demonstration of phosphatase acid in leukocytes (Sigma Chemical Co., St. Louis, MO) were both used periodically to determine that> 98% macrophages were present in the isolated, adhered cell populations.
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