Imark microplate reader

ScFvs were diluted from 4 μM to 1.28 nM in a 1:5 dilution ratio. The ELISA was performed in 96-well plates coated with scFvs (100 μL/well) at 4 °C overnight. Each well was added with Aβ42 to 3 μM. After incubating at 37 °C for 1 h, the plates were washed three times with PBST. β-Amyloid (B-4) (1:2000) was added to the wells for 2 h at 37 °C. After washing with PBST, HRP-labeled goat anti-human IgG (H+L) (1:4000) was added into each well. After incubating at 37 °C for 1 h, 3,3′,5,5′-tetramethylbenzidine solution (Tiangen Biotech, Beijing, China) was added to develop color, and color development was stopped using 2 M H₂SO₄. The absorbance at 450 nm was measured using an iMarK™ Microplate Reader (Bio-TEK, Winooski, VT, USA).

The Eucommia ulmoides Oliver bark extracting solution was freeze-dried to obtain the extract. The antiproliferation of Eucommia ulmoides extracts was tested on CT26 and HepG2 cell lines for toxicity. Stock solutions of Eucommia ulmoides Oliver bark extracts were diluted to 1, 2, 4, 6, and 8 mg/ml. Each dilution of 100 μl was transferred into the 96-well-plate when the confluency reached 60%. A blank control was also performed at the same time. The plate was placed in the MCO-15AC carbon dioxide incubator under the condition of 37°C, 5% CO₂. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect the effects of eucommia ulmoides extract on CT26 and HepG2 cell viability. The 96-well-plate was read at 490 nm after being shaken with the iMark Microplate Reader (BioTek, USA). Three replicate tests were carried out and the average values were reported.