Universal microplate spectrophotometer

ECs and SMCs were grown on transwell filters until cultures reached 90% confluency. Complete media was then removed and replaced with complete media lacking CS. To find proper magnitude and duration of LSS to build a cell model with a pathological state, the samples were then transferred to the flow chamber and incubated at 37 °C for 6, 12, or 24 h under shear stresses of 1, 2, 4, 6, 8, or 10 dyn/cm², respectively. As a control, ECs and SMCs were grown in the absence of LSS (static control cells) for 6, 12, or 24 h, respectively. EC or SMC cell suspensions were collected from either side of the transwell filter and centrifuged (1000 rpm, 5 min, 25 °C), and the cells were uniformly resuspended in the complete medium. A cell counting kit-8 (CCK8) (Beyotime, Shanghai, China) was used to detect the cell viability. Briefly, the CCK8 reagent was added in the cell suspensions in 96-well plates (20 μL/well), and the cells were maintained in the humidified cell culture chamber for 1 h. Then, the optical density (OD) of the samples was determined at 450 nm by a universal microplate spectrophotometer (Bio-Rad). Cell viability can be calculated according to the following formula: Cell Viability (%) = OD_EG/OD_CG × 100%, where OD_EG represents the OD value of experiment group and OD_CG represents the OD value of control group.

All mice were anesthetized and decapitated after behavioral experiments immediately. Hippocampus and cortex were carefully dissected from brains for examination. The hippocampus and cortex were carefully dissected from brains for examination. All the processes were performed on ice-cold plate. Tissues were rapidly stored at – 80°C. The hippocampus and cortex tissues were homogenized with ice-cold saline. The homogenate was centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was collected and the total protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Nianjing Jiancheng Bioengineering Institute, Nanjing, China) for the assay of the AChE and ChAT activities and measuring the level of ACh. Then the supernatant was used to detect the Ach concentration and the activities of ChAT and AChE according to the manufacturer's instructions by using Universal Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA).

The hippocampus and cortex tissues were homogenized with ice-cold saline and centrifuged at 12,000 ×g for 10 min at 4°C. The supernatant was used to detect the levels of MDA and the activity of SOD according to the manufacturer's instructions by using Universal Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA).