Normal mouse serum

We purchased the following tissue culture reagents from Life Technologies (Carlsbad, CA): RPMI 1640, Phenol Red-free RPMI 1640, Dulbecco’s Modified Eagle Medium, fetal calf serum, GlutaMAX, sodium pyruvate, Non-essential amino acids, penicillin/ streptomycin, Hank’s buffered saline solution (HBSS), nigericin, valinomycin, fluorescein dextran (average molecular weight 3,000 Daltons), Texas Red dextran (average molecular weight 3,000 Daltons) and SYTOX red. Recombinant IFN-γ, TNF-α and M-CSF were purchased from Peprotech, Inc (Rocky Hill, NJ). Recombinant IL-4 as well as APC-cy7 conjugated anti-CD11c (clone N418), perCP-cy5.5 conjugated anti-CD45 (clone 30F11) and PE-cy7 conjugated anti-Gr-1 (clone RB6-8C5) were from Biolegend (San Diego, CA). Sabouraud Dextrose broth and agarose were purchased from BD biosciences (San Jose, CA). Normal mouse serum was from Innovative Research (Novi, MI). Fluconazole was purchased from Sigma-Aldrich Corporation (St. Louis, MO). Uvitex-2B was purchased from Polyscience, Inc (Warrington, PA). Glass bottom culture dishes (catalog # P35G-1.5-14-C) were from Mat-tek Corporation (Ashland, MA). Dextran doubly-conjugated with fluorescein plus sulfarhodamine 101 (average molecular weight approximately 10,000 Daltons) was a custom product purchased from TdB Consultancy (Uppsala, Sweden).

Lenses (n = 4) were dissected from 10-week-old Tg2576 transgenic (Tg⁺) mice and age-matched non-transgenic (Tg⁻) littermate control mice (n = 4). One lens from each mouse was placed in a separate well containing tissue culture media (M199; Sigma-Aldrich, Burlington, MA) supplemented with 1% penicillin/streptomycin (Invitrogen/Thermo-Fisher Scientific, Waltham, MA) and 10% heat-inactivated normal mouse serum (Innovative Research, Southfield, MI). Cultures were incubated with humidification, 5% carbon dioxide, at 37 °C (Sanderson et al., 2000 (link)). Samples of conditioned media from cultures containing explanted intact lenses were collected after 2 days and 6 days of incubation. Media collected from cultures incubated without dissected lenses served as a negative control. Quantitative measurement of human Aβ species was performed on conditioned and control media samples using an anti-human Aβ fluorescent enzyme-linked immunosorbent assay (BioSource, Invitrogen, Carlsbad, CA).