Human β amyloid 1 40 elisa kit 2

Initial measurements of Aβ40 and Aβ42 content of 100 µg total solubilized rat brain lysate was done with Human β Amyloid (1-42) ELISA Kit – High Sensitive (Wako) and Human β Amyloid (1-40) ELISA Kit II (Wako). Absorbances at 450 nm were read on an xMark Spectrophotometer (Biorad).
For analysis of Aβ and sAPPs, the following Meso Scale Discovery kits were used: Aβ38, Aβ40, and Aβ42 were measured with V-PLEX Plus Aβ Peptide Panel 1 6E10 (K15200G) and V-PLEX Plus Aβ Peptide Panel 1 4G8 (K15199G), sAPPβ-Sw was measured with sAPP Swedish sAPPβ (K151BUE), and sAPPα/β-WT were measured with sAPPα/sAPPβ (K15120E), according to the manufacturer’s recommendations. Plates were read on a MESO QuickPlex SQ 120. Data were analyzed using Prism software and represented as mean ± SEM.

iPSCs were seeded at a density of 1.0 × 10⁵ cells/well in a 48-well plate and cultured in neuronal medium with 2 µg/ml Dox and 2 µg/ml Y27632 at 37 °C in 5% CO₂. Five days after seeding the cells, the medium was replaced with fresh neuronal medium, and cells were cultured until day 20 or day 40. The culture medium was fully changed with 500 µl/well of fresh medium 72 h before the harvest. The culture supernatant was centrifuged at 200× g to remove insoluble material and the supernatants were collected and stored at −80 °C. The remaining neuronal cells were lysed in RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA), and the protein concentration was measured by BCA Protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Aβ40 and Aβ42 levels in the conditioned medium were measured using the Human β Amyloid (1–40) ELISA Kit II (FUJIFILM Wako Pure Chemical, #298–64601, Osaka, Japan) and Human β Amyloid (1–42) ELISA Kit High Sensitive (FUJIFILM Wako Pure Chemical, #296–64401, Osaka, Japan), according to the manufacturer’s protocol. The concentration of each Aβ species was normalized by the protein levels in the culture.

iPSC-derived neurons were differentiated by plating almost the same number of NPCs and cultures were maintained in 48-well plate until analysis (day 45). Medium was fully changed with 500 μl/well of fresh medium 48 h before the harvest. The collected medium was centrifuged to remove insoluble material and stored at −80°C until analysis. The remaining neuronal cells were lysed in RIPA buffer and protein concentration was measured by BCA Protein assay (Pierce). Aβ40 and Aβ42 levels in the conditioned medium were measured using commercial kits, Human βAmyloid (1–40) ELISA kit II (catalog #298-64601) and Human βAmyloid (1–42) ELISA Kit High Sensitive (catalog #296-64401) from Wako, respectively, according to the manufacturer’s protocol. Each Aβ concentration was normalized by protein levels of the culture.