2.2. BV2 Cell Culture and Treatment

Mouse BV2 microglial cells (obtained from Dr. Grace Y. Sun (University of Missouri at Columbia, MO, USA) were routinely grown in low-glucose (5 mM) DMEM (Thermo Fisher/Gibco, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS) (Gibco) and antibiotics (Gibco) at 10% CO₂. For all of the assays unless otherwise noted, the cells were plated at 5 × 10⁵ cells in 35 mm tissue culture dishes. After overnight growth, the cells were transferred to high-glucose (25 mM) DMEM supplemented with 10% FCS and treated with 25 ng/mL LPS alone or in the presence of the different compounds which were added 30 min before the treatment with LPS. After 24 h, the medium was removed, spun briefly to remove floating cells and 100 µL was assayed for nitrite, the stable oxidative end product of NO and therefore a measure of its production, using 100 µL of the Griess Reagent (Sigma Aldrich) in a 96 well plate. After incubation for 10 min at room temperature, the absorbance at 550 nm was read on a Molecular Devices microplate reader. The remaining culture supernatants were stored at −20 °C until used to determine their levels of IL6 and TNFα using ELISAs (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. In order to control for differential effects of the various treatments on cell survival, the cells left in the dishes after removal of the culture supernatants were assayed for viability using the MTT assay as described previously [25]. NO or cytokine production was normalized to the MTT assay results. Because the absolute levels of NO or cytokine production varied somewhat from experiment to experiment, in order to compare the results from different experiments, the levels were normalized to the level seen with LPS alone. Absolute levels of nitrite ranged from 8 to 12 µM, IL6 from 400 to 700 ng/mL and TNFα from 300 to 500 ng/mL in the different experiments. In all cases, the absolute levels of nitrite, IL6 and TNFα in the untreated BV2 cells were minimal.