Enzyme labeled instrument

The human pancreatic cancer cell line MIA-PaCa-2 was cultured to logarithmic growth phase, and a single-cell suspension was prepared using trypsin (Solarbio Life Science, Beijing, China). A counting plate was used to regulate the cell density, and 5 replicate wells per group with 5000/100 μl plates were set up in 96-well plates. To reduce the effects of evaporation, PBS buffer was included in the edge wells prior to overnight incubation. Taxifolin was diluted to the target concentrations (2. 5, 5, 10, 20, 40, 80, 160 μmol/L) the following day. The DMSO concentration in the 160 μmol/L Taxifolin was utilized as a negative control. The liquid in the plate was removed. DMSO and Taxifolin were then given to each of the wells at the above concentrations. A control group was included as a reference. The plates were subsequently incubated for 48 hours (h). Next, 10 μL of CCK-8 solution (Toho Chemical, Japan) was given to each hole in the plates, which were returned to the incubator for one hour. After incubation, we measured the Optical Density (OD) at 450 nm using an enzyme-labeled instrument (Molecular Devices, Shanghai, China). The obtained results were subsequently analyzed and processed.

The cells in the exponential phase were inoculated in 96-well culture plates (4,000/well) and 200 μl DMEM containing 10% FBS was added, with six duplication wells for each group and an empty well (without cells) as a control. CCK-8 (20 μl) was added to each well. Following incubation for 4 h, the absorbance value at 490 nm was detected using an enzyme-labeled instrument (Molecular Devices, Sunnyvale, CA, USA). A growth curve was then plotted using the average absorbance value of the cells against the time points of 48, 72, 96 and 120 h.

The cells were seeded in 96-well plates at the density of 1 × 10⁵ cells per well for 72 hours, and then 50 μg MTT was added to each well. After shaking and mixing, the cells were cultured in a 37°C incubator including 5% volume fraction of CO₂ for 4 hours. After incubation, the supernatant was removed, and 100 μl DMSO (from Sigma-Aldrich) was added into every well and oscillated for 10 min to completely dissolve. The absorbance of each hole was determined at 490 nm by using the enzyme-labeled instrument (from Molecular Devices). Cell proliferation rate (%) = A (propofol)/A (control) × 100%.

NO content was assessed using an NO Assay Kit (Beyotime), according to the manufacturer’s instructions. HUVECs were lysed on ice with radioimmunoprecipitation assay (RIPA; Beyotime) buffer and then centrifuged. The supernatant was collected and added into the 96-well plates at 50 μl/well. Griess Reagent I was first added to each well at room temperature, followed by Griess Reagent II at room temperature. Finally, absorbance was measured at 540 nm with an enzyme-labeled instrument (Molecular Devices, LLC).

Serum levels of TC, TG, LDL-C, and HDL-C were analyzed via an enzyme-labeled instrument (Molecular Devices Co., Ltd. California, United States). The serum levels of IL-1β, IL-6, TNF-α, ALOX5, PTGS2, VEGFA, and eNOS were determined using ELISA kits following the manufacturer’s instructions.