Flat black 96 well microfluor plates

NO assays were performed as described previously.^{6 (link)} BV-2 microglia cells were grown in DMEM medium supplemented with 10% FBS. BV-2 cells were detached from the flask by trypsin digestion when ~80% confluence was reached. Cells were seeded at a density of 4×10⁴ cells/well in 96-well plates. After overnight incubation, the medium was aspirated and changed to DMEM (without FBS). LPS (200 ng/mL) and indicated concentration of compounds were added. Following an additional 24 h treatment, 100 μL of media was removed and added to flat black 96-well microfluor plates (Thermo Scientific, MA, USA). Subsequently, 10 μL of 2,3-diaminonaphthalene (0.05 mg/mL in 0.62 M HCl) was added to each well and incubated for 15 min. The reaction was quenched by addition of 5 μL of 3 M NaOH and the plate was read on a Beckman Coulter DTX880 reader (Fullerton, CA, USA) with excitation at 360 nm and emission at 430 nm. Assay of NO in BV-2 microglia cells, rather than assay of HEK293 TLR4 over-expressing cells, was chosen as the HEK293 TLR4 cells can only report activation of NFkappaB signaling pathways so do not appropriately represent the complexity of TLR4 signaling.^{37 (link), 38 (link)}

Raw 264.7 cells (mouse leukemic monocyte macrophage cell line) were grown in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml), seeded in 96-well plates at 80,000 cells per well, and grown for 24 hours at 37°C in a 5% CO₂ humidified incubator (27 (link)). After 24 hours, nonadherent cells and medium were removed and replaced with fresh unsupplemented RPMI 1640 medium. The adherent macrophages were treated with 66 nM (100 ng/ml) Pam₃CSK₄ (InvivoGen) or different concentrations of CU-T12-9. Plates were then incubated for an additional 24 hours. After incubation, 100 µl of medium was collected and added to flat black 96-well microfluor plates (Thermo Scientific). To each well, 10 µl of 2,3-diaminonaphthalene (0.05 mg/ml in 0.62 M aqueous HCl solution) was added and incubated for 15 min in the dark. The reaction was quenched by addition of 5 µl of a 3 M aqueous NaOH solution, and the plate was read on a Beckman Coulter DTX 880 reader with excitation at 365 nm and emission at 450 nm. The nitrite (a stable metabolite of NO) concentration was determined from a nitrite standard curve (48 (link)).