Griess kit

BV2 cells (4.0 × 10⁵ cells/well) or rat primary microglia (1.0 × 10⁶ cells/well) were cultured in 6-well plates coated with PLL and then incubated for 24 h with the indicated treatment. NO levels in the culture supernatants were determined using a Griess kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The absorbance was measured at 540 nm on a microplate reader.

Bone marrow-derived macrophages (BMDMs) were generated by flushing of murine tibia and femur bones with sterile PBS and culturing of bone marrow cells in complete RPMI-1640 medium supplemented with 50 μM β-mercaptoethanol (Sigma-Aldrich, Rehovot, Israel). Cells were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF) (20 ng/ml, Peprotech, Rocky Hills, NJ, USA) for 6 days of culturing, as described (59 (link)). Cells were routinely verified to be >90% F4/80⁺. For activation experiments, cultured BMDMs were seeded at 2 × 10⁵ cells per well in 200 μl of standard RPMI-1640 medium and pretreated with hAAT at 0.5 or 2.5 mg/ml, as indicated, S-NO-AAT at 2.5 mg/ml or GSNO (Chem-Impex International, Wood Dale, IL, USA) at 27.5 μM. After 24 h, cells were carefully washed with PBS and activated with recombinant IFNγ (10 ng/ml, R&D Systems, Minneapolis, MN, USA) and IL-1β (10 ng/ml, Prospec, East Brunswick, NJ, USA), with or without coculturing with RMA cells at a ratio of 1:2 (RMA:BMDM) for 24 h. IL-6 release to the supernatant was quantified with specific ELISA (Biolegend, San Diego, CA, USA). Lactate dehydrogenase (LDH) release to the supernatant was quantified with a Non-Radiological Cytotoxicity Kit (Promega, Madison, WI, USA). Nitric oxide release to the supernatant was evaluated by Griess kit (Promega) according to the manufacturer’s instructions.

Nitrite release was measured as an indicator of NO production. BV2 cells (4.0 × 10⁵/well) or primary rat microglia (1.0 × 10⁶/well) were seeded in 6-well plates pre-coated with PLL and incubated for 24 h with the indicated drugs. The nitrite concentration in the culture supernatant was evaluated with a Griess kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The absorbance at 540 nm was measured on a microplate reader (Elx800; Bio-Tek Instruments, Winooski, VT, USA).