Varioskan flash instrument

Cell proliferation was assessed with Cell Counting Kit‐8 (CCK‐8) (Dojindo Lab, Japan) according to the manufacturer’s instructions. In brief, a total of 5000 cells/well were seeded into 96‐well plates, and then, cells were cultured in the indicated conditions for 72h, and the proliferation index at 450 nm absorbance was measured by a Thermo Scientific Varioskan Flash instrument after incubation with 10 µl CCK‐8 solution for 2 h at 37 °C. Cell proliferation was also assessed via IncuCyte ZOOM (Essen BioScience). A total of 5000 cells/well were seeded into 96‐well plates and were automatically monitored and recorded every 2 h by the IncuCyte ZOOM for 72h.

Cells cultured in 24‐well plate were collected and incubated with calcein‐AM (2 μM) in a medium containing 120 mM NaCl, 5.0 mM KCl, 2.0 mM CaCl₂, 20 mM HEPES, and 15 mM glucose (pH 7.4) at 37°C for 30 min, which causes penetration into the cytoplasm and mitochondria. The fluorescence from cytosolic dye was quenched for another 30 min by the addition of CoCl₂ (1 mM). After washing cells three times, half of the cells were left as is, and the other half were treated for another 30 min with 500 μM CaCl₂, Ca²⁺ ionophore calcimycin (5 μM), CATR (1 μM), or BKA (5 μM) to trigger or inhibit mPTP opening. Then, the calcein fluorescence was detected by the Varioskan flash instrument (Thermo Scientific, USA) and the decreased percentage of initial calcein fluorescence could be accepted as the mPTP opening level.

The cell proliferation inhibitory effect of UA-PMs was performed against human hepatocellular carcinoma cell line HepG2 and human normal liver cell line L-02 by MTT assay. All cells were seeded into 96-well cell plates with 1 × 10⁴ cells/well and incubated for 24 h until reaching confluence. Then, they were treated with free UA and UA-PMs at different concentration of 2.5, 5, 10, 20, 30, 40, 50, 60, 80, 100 μmol/L using DMEM medium as negative control and 5-FU as positive control. After incubation for 24 h, 20 μL of MTT solution (5 mg/mL) was added and incubated for another 4 h in the dark. The supernatant medium was removed and the left MTT-formazan crystals were dissolved in 150 mL DMSO. Then the plate was incubated for 10 min at 37 °C with gentle shaking before determining the absorbance at 490 nm using Varioskan Flash instrument (Thermo Fisher, Waltham, MA, USA). The inhibitory rate of cell proliferation was obtained through the following formula as: Inhibition Rate (%) = (1 - A_sample / A_control) × 100%. Each assay was conducted in triplicate.

IEB was pretreated with or without the inhibitor of AQP1 channel, 10 μM bacopaside II, for 1 h, which was followed by the addition of 4 kDa FITC-conjugated dextran (1 mg/mL) to the upper chamber of Transwells. After 2 h, 4 h or 6 h incubation, 100 μL of sample was collected from the lower chamber to quantify the amount of FITC–dextran penetrated from the upper to lower chamber. Meanwhile, 100 μL DMEM was supplemented into the lower chamber. Fluorescence was read using a VARIOSKAN FLASH instrument (Thermo Scientific, Waltham, MA, USA) that detects the fluorescence at excitation and emission wavelengths of 495 nm and 519 nm, respectively. The values of the samples were expressed as fold increase in the fluorescence intensity in mock-infection.

The granulosa layers of F5 were used for protein isolation with the BSP003 kit (Sangon Biotech Co., Ltd, Shanghai, China). Protein concentration was measured with the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA) and Varioskan Flash instrument (Thermo Fisher Scientific, Rockford, IL, USA). A total of 30 μg protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in 1× Tris-Buffered Saline with Tween (TBST) buffer for 1 h at room temperature, membranes were incubated with rabbit anti-chicken (ERα, ERβ, PR-A, and PR-B) monoclonal antibodies (Abcom, Cambridge, UK, 1:1000) and rabbit anti-chicken β-actin monoclonal antibody (Abcom, Cambridge, UK, 1:1000) overnight at 4°C [27 (link)–29 (link)]. The blots were then washed in 1× TBST buffer and probed with goat-anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody (diluted 1:2000 in 1× TBST; Abcom, Cambridge, UK) for 1 h at room temperature. Binding was visualized with enhanced chemiluminescence (ECL) reagent (Beyotime Institute of Biotechnology, Jiangsu, China) using a ChemiDoc XRS instrument (Bio-Rad, Inc., Richmond, CA, USA). Quantity One Software (Bio-Rad, Inc., Richmond, CA, USA) was used for densitometric analysis.