Infinite m1000 pro multiscan spectrum

Cell survival was measured by cell counting kit-8 (CCK-8) purchased from Dojindo (Kumamoto, Japan). 1×10⁴ BV2 cells, 2.5×10⁴ primary microglial cells, 2.5×10⁴ primary astrocytes and 5×10⁴ primary neurons were seeded in 96-well plates, respectively, and treated with RRx-001 at different concentrations with or without LPS (100 ng/ml) after cell adherence. After 24 h, the cells were incubated with 100 µl CCK-8 working solution for 2 h at 37°C. The absorbance was evaluated at 450 nm by using the infinite M1000 PRO multiscan spectrum (Tecan, Switzerland).

1×10⁴ BV2 cells were seeded in 24-well plates, and pretreated with RRx-001 at proportionate doses of 0.2, 1 and 5 μM and 5×10⁴ primary microglial cells at doses of 0.2, 0.4 and 0.8 µM for 12 h, followed by LPS (100 ng/ml) for 24 h. After the cultured media were collected, the levels of TNF-α and IL-6 were measured by using a mouse TNF-α ELISA kit (Dakewe, Beijing, China) and mouse IL-6 ELISA kit (Boster Biological Technology co. ltd., Wuhan, China). Adhered cells were collected with trypsin and ultrasonicated to detect Nox4 activity with mouse NOX4 ELISA kit (Reddotbiotech, Kelowna, Canada). The absorbance was evaluated at 450 nm by using the infinite M1000 PRO multiscan spectrum (Tecan, Switzerland).