Aβ1 40

Aβ_1–42 and Aβ_1–40 were purchased from Peptide Institute Inc. (Osaka, Japan), and SH-SY5Y cells (human neuroblastoma; EC94030304) were obtained from the European Collection of Authenticated Cell Cultures (London, UK). Dulbecco’s Modified Eagle Medium (DMEM) Ham’s/F-12 medium and all-trans retinoic acid (ATRA) were obtained from Fuji Film Wako Pure Chemical Co., Ltd. (Osaka, Japan). Penicillin G sodium, streptomycin sulfate, amphotericin B, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific K.K. (Waltham, MA, USA). 17β-estradiol and G-15 were procured from Cayman Chemical Company (Ann Arbor, MI, USA), and raloxifene hydrochloride was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The other chemicals used in this experiment were commercially available and of the purest grade. Their structures are shown below (Figure 1). Special-grade products were used for other reagents.

A solid form of Aβ(1–40) was purchased from the Peptide Institute (Osaka, Japan). Aggregation was carried out by gently dissolving the peptide (0.50 mg/mL) in phosphate-buffered saline (PBS) (pH 7.4). The solution was incubated at 37 °C for 42 h with gentle and constant shaking. A mixture containing 50 μL Aβ(1–40) aggregates (final conc., 1.25 μg/mL), 50 μL ^99mTc-Ham complex (final conc., 8.3 kBq/mL), 50 μL PIB (final conc., 64 pM–125 μM in 30% EtOH), and 850 μL of 30% EtOH was incubated at room temperature for 3 h. The mixture was filtered through Whatman GF/B filters (Whatman, Kent, U.K.) using a Brandel M-24 cell harvester (Brandel, Maryland, USA), and the radioactivity of the filters containing the bound ^99mTc-Ham complex was measured using a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, USA). Values for the half-maximal inhibitory concentration (IC₅₀) were determined from displacement curves using GraphPad Prism 5.0 (GraphPad Software, Inc., California, USA).

Human CRP and SAP were isolated by the team of Professor Mark B. Pepys (Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London) as described elsewhere53 (link)54 (link)55 (link). CRP and SAP were obtained with full informed consent from each individual donor. All individuals were paid donors in the USA, where the plasma was collected at centers approved by the UK Department of Health. Donor selection, donor examination and plasma collection were performed according to standards and/or requirements set by the UK Department of Health, in accordance with the European Pharmacopoeia monograph ‘Human Plasma for Fractionation’. No additional or specific ethical committee approvals were sought as these are not required for experimental in vitro use of plasma proteins isolated from donor plasma obtained with informed consent and processed under the prevailing strict regulatory guidelines specified here. AGP and GST were obtained from Sigma. MDH and LDH were obtained from Roche. Aβ(1-40) was purchased from Peptide Institute, Inc. (Osaka, Japan).

ARPE-19 cells (ATCC® CRL-2302™), a human RPE cell line [10 (link)], were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagles medium (Gibco®, Life Technologies, Carlsbad, CA) and F12 medium (Gibco®) 1:1 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin G, and 100 μg/mL streptomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To examine the effects of Aβ on the ARPE-19 cells, several concentrations of Aβ 1–40 (Peptide Institute, Inc., Osaka, Japan) were added to the culture medium. Equivalent amount of DMSO (Wako Pure Chemical Industries), a solvent for Aβ1–40, was added to the culture medium in the control group.