In nite f200 pro microplate reader

Nitrite of microglia supernatant was quanti ed by Griess reaction colorimetric test. Microglia culture media was collected and centrifuged to remove cellular debris and subsequently incubated with Griess reagent (1:1 ratio, Sigma-Aldrich) for 10 minutes at room temperature (RT), protected from light. Absorbance was measured at 540 nm using an In nite F200 PRO microplate reader (Tecan). Nitrite concentration was calculated with reference to a standard curve generated with known concentrations of sodium nitrite (NaNO 2 , Sigma-Aldrich).

Enzyme-linked immunosorbent assay (ELISA) was performed to measure Tumour necrosis factor α (TNFα), Interleukin-1 β (IL-1β) and IL-10 microglia supernatant levels, using the respective Standard ABTS ELISA Development Kits (PeproTech). For Glial cell-derived neurotrophic factor (GDNF) and Brain-derived neurotrophic factor (BDNF), different kits were used (GDNF ELISA Kit (Abcam), Human/Mouse BDNF DuoSet ELISA (R&D)). Microglia culture media was collected and centrifuged to remove cellular debris, and the pellet was discarded. The resulting supernatant was kept at -80°C until analysis. All experiments were performed in accordance with the respective manufacturer's instructions. Absorbance values were measured at 415nm, with wavelength correction set at 560nm, using an In nite F200 PRO microplate reader (Tecan).