ABSTRACT
BM are harvested from bronchoalveolar saline lavages of adult male rats. They are tested for viability with trypan blue (>90% viable) and transferred to 96-well microplates (100,000 cells per well) containing culture media. The cells are incubated in 5% CO2 at 37°C for up to 5 hours to achieve attachment. The culture media is replaced with media containing a test compound or mixture and incubated for up to 1 h. At the end of the incubation period, the media is removed and stored at −20°C for assay of lactate dehydrogenase (LDH) and TNFα. To measure phagocytic oxidative burst, zymosan and luminol containing media is added to the cells and the resultant chemiluminescence over 1 h is measured with a FluoStar micoplate reader (BMG Labtechnologies, Durham, NC).
