Aβ40 and aβ42 elisa kits

Conditioned media from HEK cells transfected with WT or mutant APP was assayed for Aβ by 4G8 or 6E10 Aβ ELISA kits from Meso Scale Diagnostics. Aβ levels were measured by both 6E10 and 4G8 ELISA for each data point, yielding nearly identical results. In vitro assay Aβ was measured using Aβ40 and Aβ42 ELISA kits from Invitrogen. All ELISAs were performed according to the manufacture’s protocols.

Aβ ELISA was performed as previously described [23, 24] (Liu et al., 2014 (link); Fang et al., 2016 (link)). Briefly, cortical and hippocampal tissues were homogenized in Tris-buffered saline (TBS) containing a protease inhibitor cocktail (Sigma) and then centrifuged at 16,000 ×g for 30 min at 4°C. The supernatant (TBS-soluble fraction) was collected, and the pellets were homogenized in TBS supplemented with 1% Triton X-100 (TBST) containing a protease inhibitor cocktail (Sigma), sonicated for 5 min at 4°C in a water bath, and then centrifuged at 16,000 ×g for another 30 min at 4°C. The supernatant (TBST-soluble fraction) was collected, and the pellets were extracted a third time with an ice-cold guanidine buffer (5 M guanidine HCl/50 mM Tris, pH 8.0) and referred to as the guanidine-soluble fraction. The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Biotechnology). The Aβ concentrations were detected by Aβ40 and Aβ42 ELISA kits (Invitrogen) following the manufacturer’s instructions. The levels of Aβ were normalized to the amount of total protein in the tissues.

Protein expression levels were determined using Western blot analysis. The protein samples were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (BIO-RAD, United States), which were then incubated overnight at 4°C in the following primary antibodies: anti-PS1 (Thermo Fisher Scientific, cat. no. PA5-98093, United States), and anti-GAPDH (CST, cat. no. 5174, United States). Three independent experiments that included both technical and biological replicates were performed. To explore Aβ levels, Aβ40, and Aβ42 ELISA kits (Invitrogen, United States) were used according to the manufacturer’s instructions. In brief, the cell media were mixed with Aβ 40-, or Aβ42-specific antibody and shake-incubated in a 96-well plate overnight at 4°C. After incubation with secondary antibody for 30 min, the reaction with substrate was carried out at room temperature. The color intensity was measured at 450 nm using a multimode plate reader (EnVision^®, Perkin Elmer, United States). Five independent experiments that included both technical and biological replicates were performed.

The Aβ42 and Aβ40 levels in 100-μl samples and the corresponding standards were detected using ELISA with Aβ42 and Aβ40 ELISA kits (Invitrogen Corporation, Camarillo, California, USA). At 450 nm, the absorbance was read by a Multiskan Spectrum Microplate Spectrophotometer (Thermo Fisher Scientific Oy Ratastie 2, Vantaa, Finland), which was used to quantitate the Ms Aβ 40 or Ms Aβ 42 concentrations according to the standard curve plotted. The Aβ42 and Aβ40 levels were subsequently standardized and expressed as pg/mg (Aβ42 or Aβ40 mass/hippocampus tissue weight).