Human rat β amyloid 40 elisa kit

We measured Aβ40 and Aβ42 using cell supernatants conditioned for four days. We performed three biological replicates. Supernatants were collected at different time points, and frozen at − 80 °C. Secreted Aβ40 and Aβ42 were measured using Human/Rat β Amyloid (40) ELISA Kit (Wako) and Human/Rat β Amyloid (42) ELISA Kit (Wako, Japan), according to the manufacturer’s instructions.

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.

Blood was collected from mice under a fed condition. The insulin concentrations in the sera were determined by ELISA (Mercodia Ultrasensitive Mouse Insulin ELISA, Mercodia AB). The serum concentrations of β-amyloid 40 and 42 were quantified using a Human/Rat β-Amyloid (40) ELISA kit (WAKO) and Human/Rat β-Amyloid (42) ELISA kit (WAKO).