Hilyte fluor 488

Fluorescent (HiLyte^TM Fluor 488) labeled Aβ42 was purchased for cell culture assays from Anaspec (HiLyte™ Fluor 488 –DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). This peptide had a molecular weight of 4870.5 g/mol, absorption/emission wavelengths of 503/528 nm, and was judged to be > = 95% pure by HPLC (CAT# AS-60479, LOT# 1958003). To prepare the Aβ peptide for cell culture assays, 0.1mg of the peptide was dissolved in 30 uL of 1% (w/v) ammonium hydroxide in sterile water and filtered using a 0.45 um pore filter [81 (link)]. Once thoroughly dissolved and mixed, 380 uL of Phosphate Buffered Saline (PBS) solution were added to the peptide-ammonium hydroxide solution (final NH₄OH 0.073%). After mixing thoroughly again, the peptide solution was aliquoted into 50 μL aliquots and frozen until use. When using the peptide solution for cell culture experiments, the peptide containing aliquot was thawed and vortexed immediately before use.

Human Aβ (1–42), unlabeled and labeled with HiLyte™ Fluor 488 (ex/em 503/528 nm), was purchased from AnaSpec Inc. (Seraing, Belgium). This was a mixture of monomers and amorphous aggregates of various size and shape [23 (link)]. The Aβ was dissolved in deionized water (500 µM) and frozen in aliquots at −80 °C for further use.

Aβ (1–40) and Aβ (1–42) peptides, labeled with HiLyte™ Fluor 488 or HiLyte™ Fluor 647 at the N-termini, were purchased from AnaSpec (BioNordika, Solna, Sweden). Aβ peptides were reconstituted by adding 40–50 µl 1% NH₄OH to 0.1 mg dry peptide and diluted to 1 mg/ml with PBS, then aliquoted and stored at −20 °C.

Coenzyme Q10 was generously provided by Kaneka Corporation. Human Aβ_25–35 and tempol were purchased from Tocris. Human Aβ_25–35 labelled with HiLyte Fluor 488 was obtained from Anaspec. The fluorescent probes, Fluo-4 AM, H₂DCF-DA, MitoSOX-AM, MitoTracker Deep Red, Calcein-AM and Hoescht were obtained from LifeSicences. Alpha tocopherol was acquired from Sigma-Aldrich.

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.

To investigate the roles of EPB41L4A-AS in Aβ clearance, EPB41L4A-AS shRNA cells and NC shRNA cells were inoculated with Aβ_1-42, HiLyte Fluor™ 488-labelled (ANASPEC, AS-60479-01). After 0, 12, 24, and 36 hours, the cells were washed, fixed, and counterstained with DAPI and observed under an Olympus FV1000 confocal laser microscope.