All Aβ measurements were performed using ELISA kits listed in Key Resources Table according to the manufacturer’s instructions. Cultured neurons were washed 3 times on D10 with NBA/B27 to remove existing Aβ, and treated with control solution or ApoE2, ApoE3, or ApoE4 (10 µg/ml in NBA/B27, 0.5 ml per well on a 24-well plate) for 48 hours. The culture medium was harvested at D12 to examine the concentration of secreted Aβ variants. The cultured neurons were then washed for 3 times with PBS, and the wash buffer from the last wash was collected and analyzed for complete removal of ApoE and soluble Aβ by immunoblotting of ApoE and Aβ ELISA. After the washes, lysis buffer (TBS, 1% Triton X-100, 50 µl per well) was added, and the levels of cellular Aβ extracted in cell lysates were measured by ELISA. The HEK293 ApoE proteins (medium supernatants from ApoE-expressing HEK293-F cultures; Methods, Recombinant ApoE and RAP proteins, A), media from mouse glial and cortical cultures were also subjected to ELISA and confirmed to be free of human Aβ.
