Hilyte fluor555

Human Aβ(1–42)-HiLyte™-Fluor555 was obtained from AnaSpec (Freemont, CA, USA). The unlabeled Aβ(1–42) were synthesized and supplied by StabVida (Caparica, Portugal) and GenicBio Limited (Shanghai, Popular Republic of China). The peptide VFAFAMAFML (amidated-C-terminus amino acid)—synthetized upon request and supplied by GenicBio Limited (Shanghai, Popular Republic of China)—was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM and stored at −20 °C until use. Purified bovine brain CaM, HisTrap^TM FF and a Thrombin Clean Cleavage^TM kit were purchased from Sigma-Aldrich (Madrid, Spain). Microcolumns NZYMiniPrep were acquired from NZYTech (Lisbon, Portugal).
All other reagents and chemicals were of analytical grade from Sigma-Aldrich (Madrid, Spain), Roche–Merck (Darmstadt, Germany), and ThermoFisher Scientific (Madrid, Spain).

Both cell lines were incubated with media supplemented with 200 nM human Beta-Amyloid (1-42) HiLyte Fluor 555 (AnaSpec, Fremont, CA, USA) or 200 nM human Beta-Amyloid (1-42) HiLyte Fluor 488 (AnaSpec), with or without thiethylperazine (MilliporeSigma) for approximately 18 h. Cells were then washed twice with phosphate buffered saline (PBS), trypsinized, and spun-down. Pelleted cells were washed once with ice cold PBS, then resuspended in 1% FBS in ice cold PBS, and kept on ice until assayed. Sorting occurred on the FACSCanto II (BD Biosciences) or the Sony SH100S (Sony Biotechnologies Inc.), and initially gated using untreated cells. Values are reported as the percentage of fluorescent cells.

Fluorescent HiLyte™ Fluor 555 labeled human Aβ₄₂ monomers (Aβ₄₂-555; #60480-01, AnaSpec, CA, USA) were aggregated into insoluble fibrils according to a well-established protocol. Briefly, the synthetic Aβ₄₂ monomers were dissolved in a 10 mM NaOH and 10× PBS (#11530486, Thermo Fisher Scientific) solution to a final concentration of 2 mg/ml. The Aβ₄₂ samples were incubated on a shaker at 1500 rpm, 37 °C for 4 days. Finally, the resulting Αβ-F were diluted in sterile 1× PBS to the final concentration of 0.5 mg/ml and sonicated at 20% amplitude, 1 s on/off pulses for 1 min (#VCX130, Vibra Cell sonicator, Sonics, CT, USA).