Griess reagent kit for nitrite determination

The effect of B1 AMCE on the level of nitric oxide in 4 T1 tumor was investigated using the Griess reagent assay. It was carried out using the Griess Reagent Kit for Nitrite Determination (Life Technologies, USA). Twenty microliter of Griess reagent containing equal volume of sulfanilic acid and N-1-napthylethylenediamine dihydrochloride was mixed with 150 μL of the nitrite-containing sample and 130 μL of deionized water in a microplate and incubated for 30 min at room temperature. Standard curve was also prepared by diluting the nitrite standard solution with deionized water to give a series of concentration between 1–100 μM. In place of the nitrite- containing sample, the standards were mixed with the Griess reagent and incubated in a similar manner. The absorbance of the sample and standards were read by spectrophotometer (Beckman Coultor, USA) at 548 nm wavelength before the nitrite concentrations corresponding to the standard plot could be evaluated.

Conditioned media from the cell samples were collected immediately before protein extraction and centrifuged at 10000×g for 10 min. Aβ40 levels in the conditioned media were determined with monoclonal and HRP-conjugated antibody-based Human/Rat β amyloid 40 ELISA kit (Wako, Osaka, Japan). Aβ40 concentrations were normalized to neuronal viability. Mouse TNF alpha ELISA Ready-SET-Go! kit (Affymetrix, San Diego, CA, USA) was used for the detection of tumor necrosis factor α (TNFα) in the conditioned media. Nitric oxide (NO) levels were determined using Griess Reagent Kit for Nitrite Determination (G-7921, Life Technologies) and normalized to neuronal viability determined by the MAP2-ABTS assay described above. All kits were used as instructed by the manufacturers. Reactive oxygen species (ROS) levels in the co-cultures were measured using fluorogenic probe 2′, 7′-Dichlorodihydrofluorescin diacetate (DCFH-DA, Sigma D6883). One hour after adding BV2 cells, samples were labeled with 120 μM DCFH-DA for 30 min. Two hours after adding the BV2 cells, 5 h-neuroinflammation treatment was started. Subsequently, cells were lysed using T-PER lysis buffer (Thermo Scientific), and fluorescence was measured using plate reader at 480 nm/530 nm.

Aβ40 and Aβ42 levels in the mouse hippocampus homogenates were determined with monoclonal and HRP-conjugated antibody-based Human/Rat β amyloid 40 ELISA kit (Wako, Osaka, Japan). Mouse TNF-α and IL-6 ELISA Ready-SET-Go! kits (Affymetrix, San Diego, CA, USA) were used for the detection of tumor necrosis factor α (TNF-α) and IL-6 in the conditioned media of WT and Akt2 KO mouse primary microglia cultures treated with LPS (200 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) and IFNγ (20 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Nitric oxide (NO) levels were determined using the Griess Reagent Kit for Nitrite Determination (G-7921, Life Technologies, Eugene, OR, USA). All kits were used as recommended by the manufacturers.