Hilyte fluor 488 labeled

Aβ1–40 and Aβ1–42 amounts were measured from the culture medium conditioned for 72 hr with ELISA according to the manufacturer's instructions (Invitrogen). DAPT (5 μM; Sigma) was added to the medium 1 day prior to the conditioning when applicable. Results were normalized to total protein concentration (Pierce BCA protein assay). Fluorochrome-conjugated Aβ1–42 (HiLyte Fluor 488-labeled, AnaSpec) was mixed with non-fluorescent fibrilized Aβ1–42 (from American Peptides, incubated at +37°C for 1 week) in a 1:15 ratio and added to the cells at a final concentration of 5 μM. Cells were incubated overnight, washed three times with PBS to remove non-internalized Aβ, and detached with Accutase. The percentage of 488-positive cells was determined with FACSCalibur (BD Biosciences). γ-Secretase activity was measured following a previously published protocol (Farmery et al., 2003 (link), Viswanathan et al., 2011 (link)). γ-Secretase inhibitor L685,458 (Sigma) was included for assay validation purposes.