This procedure provides a useful and sensitive assay for real-time detection of oxidant production by phagocytes (Antonini et al., 1994). Particle stocks were thawed at room temperature, sonicated for 10 s and vortexed for 30 s. Particle stocks were diluted in complete M199 containing 5% FBS. Total and insoluble particulate fractions were diluted to 2 mg/ml, whereas EHC-93sol was diluted to 500 μg/ml, in complete M199 cell culture medium. Particle suspensions (50 μl each mTOR inhibitor at 20, 50 and 100 μg) were added to the cell culture wells containing macrophages. The EHC-93sol was added at 5, 12.5, 25 μg/well to approach to the 20, 50, 100 μg/well mass equivalence of EHC-93tot

and EHC-93insol. The final volume in all wells was 150 μL (5% FBS, 200 μM luminol). Immediately after addition of AG-014699 nmr the particles, the plates were placed in a Dynatech ML-2350 Luminometer (Dynatech Laboratories, Chantily, VA, USA) at 37 °C and the luminescence was read every 5 min for a total of 2 h ( Fig. 1). All experiments were conducted as three to five independent replicates, on separate days and each time using freshly isolated rat macrophages. The cells from two to three rats were combined into a common and uniform pool of cells to supply all assays within each experiment. One to three technical replicate assays were conducted for each experimental condition within experiments. Following exposure to the particles for 2 h and the initial particle-induced

respiratory burst, we have assessed the responses of the cells to the Phosphoglycerate kinase respiratory burst stimulants PMA, Zymosan and LPS/IFN-γ (Fig. 1). Respiratory burst stimulant stocks were thawed at room temperature. Working stocks were prepared daily for PMA (4 μM), Zymosan (200 μg/ml), LPS (20 μg/ml) and IFN-γ (4000 IU/ml) in serum-free M199. Immediately after addition of the respiratory burst stimulants (50 μL per cell culture well), the plates were placed in

the luminometer at 37 °C. The final volume in all wells was 200 μL (3.75% FBS, 150 μM luminol). For PMA (final concentration, 1 μM), the luminescence was read every 2 s for 40 min. For Zymosan (final concentration, 50 μg/ml) and LPS/IFN-γ (final concentration, LPS 5 μg/ml, IFN-γ 1,000 IU/ml), the luminescence was read every 5 min for 5 h. In an initial experiment, freshly isolated alveolar macrophages were exposed to PMA, Zymosan or LPS/IFN-γ to assess the kinetics of the respiratory burst response and to determine the duration for which the respiratory burst response needed to be monitored during subsequent particle exposure experiments. Distinct respiratory burst response kinetics were observed upon stimulation, with Zymosan producing the highest baseline levels of respiratory burst as measured by luminescence, ∼20-fold higher (12,000 L.U.) than PMA (550 L.U.) or LPS/IFN-γ (610 L.U.) (Fig. 2). The viability of macrophages was determined in a separate set of culture plates after 2, 3, 7 and 24 h of exposure.