Brdu assay kit

Cell proliferation was evaluated by a BrdU assay kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. Briefly, pHUVECs (5×10³ cells/well) were seeded in 96-well plates overnight. Then, the cells were starved in serum-free ECM for 6 h and further cultured in 100 μL of serum-free ECM containing various concentrations of succinate for 24 h. Ten μL of a BrdU-labeling solution was added to each well for 4 h and the wells were dried, fixed, and measured the optical density at a wavelength of 450 nm. These assays were repeated in triplicate.

Isolation of primary mHSC was performed via pronase–collagenase perfusion followed by density gradient centrifugation in 13.2% Nycodenz (Axis-Shield PoC, Oslo, Norway) [33 (link)]. Purity of preparation was assessed by confirmation of vitamin A autofluorescence.
Isolated HSCs were allowed to attach for 2 h and were then stimulated with bile salts CDCA, GCDCA, TCDCA, and UDCA (Sigma-Aldrich, Darmstadt, Germany) for the time periods indicated, in the absence or presence of AG1478 (Sigma-Aldrich, Darmstadt, Germany) and PD98059 (Cayman, Ann Arbor, MI, USA). To quantify total DNA as a surrogate of cell number, HSCs were incubated with PicoGreen^® (Invitrogen, Carlsbad, CA, USA) and fluorescence signals were detected with a CytoFluor 4000 system (PerSeptive Biosystems, Framingham, MA, USA). Proliferation of HSC was quantified using a BrdU-assay kit (Roche, Penzberg, Germany) according to the manufacturer’s instructions. To quantify total cell count, HSCs, seeded in Lab-Tek II Chamber Slides (Nunc, Rochester, NY, USA), were mounted on cover slides with Vectashield mounting medium including DAPI (Vector, Burlingame, CA, USA). Slides were scanned with a Pannoramic Midi Slide Scanner (3DHistech, Budapest, Hungary) and nucles count was performed with ImageJ2 software on the complete slide (0.7 cm²).

The cells were plated at 2,000 cells/well in the complete culture medium in a 96-well plate and incubated at 37 °C, 5% CO₂. At day 3, cell proliferation was assessed using the BrdU assay kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instruction. Briefly, the cells were labeled with 10 μL BrdU for 3 h at 37 °C. The labeling medium was then removed and then fixed with 100 μL FixDenat solution. After removing FixDenat, 100 μL peroxidase-conjugated anti-BrdU antibodies were added to each well and incubated with the cells for 90 min at room temperature. After washing with PBS, 100 μL of a substrate solution [3, 3, 5, 5–tetramethylbenzidine dissolved in 15% (v/v) dimethylsulfoxide] was added to each sample for 30 min at room temperature. The absorbance at 450 nm was measured and reported.

The co-culture of the MSCs and T cells was carried out using 24-well plates (contact culture) and the Transwell system (isolated culture) in which the T cells and MSCs were physically separated by a membrane permeable for soluble factors. The inner hanging cell culture insets with 0.4-μm pore size membrane of a 24-well plate were purchased from Millipore (Billerica, MA, USA). The BM-, AT-, WJ- and PL-MSCs were seeded at 10⁵ cells/well in regular 24-well plates and 24-well Transwell plates containing α-MEM, 10% FBS and 100 U/ml penicillin/streptomycin. After 24 h, 10 μg/ml mitomycin C (MMC; Sigma) were added to inhibit MSC proliferation, and the cells were incubated for 2 h at 37°C followed by 5 extensive washes with medium. A total of 10⁵ T cells/well was added and stimulated with 10 g/ml phytohemagglutinin (PHA; Sigma) and 10 ng/ml interleukin (IL)-2 (Sigma). IL-2/PHA-activated T cells were cultured in the presence or absence of MSCs. The cultures were plated in triplicate and incubated for 5 days before the addition of 5-bromo-20-deoxyuridine (BrdU). After 18 h, proliferation was assessed using the BrdU-Assay kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions.