Microplate manager

Hydroxyproline quantification was determined by colorimetry using a hydroxyproline assay kit (MAK008, Sigma-Aldrich, St. Louis, MO, USA). It was determined by the reaction of oxidized hydroxyproline with 4-(dimethylamino)benzaldehyde (SMAB). The lyophilized collagens were dissolved in molecular grade water (0.5 mg/mL). The procedure indicated in the insert of the kit was followed. The reading was carried out in a plate reader (iMark^TM, Microplate Absorbance Reader, Microplate Manager software Bio-Rad, Osaka, Japan) at 560 nm, and the concentration of hydroxyproline in collagen samples was determined using an analytical curve [38 (link)].

The frozen portion of the dissected hippocampi from twenty 5XFAD mice were homogenized on ice, and then used for quantification of the soluble human Aβ₄₂ by ELISA at room temperature, according to the manufacture’s manual (Thermo Fisher Scientific, Waltham, Massachusetts, USA; Cat. # KHB3441). Briefly, 50 µL aliquots of tissue samples and reference standards (0–1000 pg/mL) were loaded into wells in the supplied 96-well plate, and 50 µL of Hu Aβ₄₂ detection antibody solution was added to each well. The mixtures were incubated for 3 h with shaking, then the plate was washed four times with supplied 1 × wash buffer. A total of 100 µL anti-rabbit IgG HRP was added to each well and incubated for 30 min, then the plate was washed four times with 1 × wash buffer. A total of 100 µL stabilized chromogen was added to each well and incubated for 30 min in the dark. A total of 100 µL stop solution was added to each well, and the plate was read at 450 nm absorbance with a microplate reader. The Aβ₄₂ levels were calculated by fitting to a standard curve using Microplate Manager (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and were expressed in pg/mL.

An equivalent number of cells (5.0 × 10³/well) was flat-bottomed 96-well microtiter plate and allowed to adhere for 24 h at 37°C. After washing with PBS twice, cells were incubated with fresh BEBM without growth factor and serum with or without recombinant soluble human TGF-β1 (10 ng/ml), TNF-α (10 ng/ml), or different concentrations of TWEAK (1-100 ng/ml) for 48 h at 37°C. After treatment, 10 μl Cell counting Kit 8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the 96-well plates were continuously incubate at 37°C for 2 h. Absorbance was read at 450 nm on a Microplate reader with Microplate manager (Bio-Rad Laboratories, Hercules, CA, USA).

Tetanus specific IgG in serum was tested in a pre-coated ELISA kit (XpressBio, mouse anti-tetanus toxoid IgG ELISA Assay, # IM-202, Thurmont, MD, USA). The standards, as well as all samples, were tested in duplicates on 96 wells plates. The plates were read at 405 and 450 nm on a microplate reader (Bio-Rad, model 550, Hercules, CA, USA), before being analyzed (Microplate manager, version 5.2.1, Bio-Rad). The samples were diluted 1:350 and treated according to manufacturer’s instructions including the setup of a standard curve to calculate antibody titers from OD values.

We used diethyl ether to extract CORT and T from plasma, measuring the efficiency of extraction (mean 96.0%) with tritiated corticosterone. CORT levels were then analyzed with EIA (Cayman Chemical Company,Cat. no. 500655 Ann Arbor, MI, USA). Samples were randomly distributed across 10 plates (within‐plate CV: 8.56%; between‐plate CV: 7.35%). Baseline plasma T concentration was analyzed using EIA (Enzo Life Sciences,Cat. no. ADI‐901‐65 Farmingdale, NY, USA). Samples were randomly distributed across 12 plates (within‐plate CV: 8.076%; between‐plate CV: 17.79%). Hormone levels were determined in reference to eight‐point (CORT) or nine‐point (T) standard curve using a cure‐fitting program (Microplate Manager, Bio‐Rad Laboratories Inc., Hercules, CA). Samples were corrected for extraction efficiency. Both assays have been previously validated in our laboratory.