Brdu incorporation

One part of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) solution was added to 5 parts of culture media, and the cells were incubated for 4 hours at 37°C. Absorbance (OD) was measured at 490 nm and 650 nm as a reference wavelength. Subsequently, nuclei were stained with Hoechst 33342 (Life Technologies) in the dark for 20 minutes at room temperature and washed with DPBS. Cell numbers were counted in the Operetta High-Content Imaging System (PerkinElmer, Rodgau, Germany) at 380 nm excitation and 445 nm emission. Cell survival rate after AcS is depicted as the percentage of initially plated cells. Cell survival rate is depicted as the percentage of cells counted after AcS, as compared to the percentage of cells plated. BrdU incorporation was used (Roche, Mannheim, Germany) to quantify cell proliferation. Cells cultured in 96-well plates were incubated with BrdU labeling solution at 37°C for 4 hours, and the following steps were performed without interruptions according to the manufacturer's instructions. Absorbance was measured in the ELISA reader (Molecular Devices GmbH, Biberach an der Riss, Germany) at 370 nm with the reference wavelength set to 492 nm.

Cell proliferation was measured using BrdU incorporation (Roche, Indianapolis, IN). Briefly, cells were incubated with 10 μM 5-bromo-2’-deoxyuridine for 4 h, and then were fixed with FixDenat solution. Anti-BrdU-POD was added to the fixed cells and substrate solution containing tetramethylbenzidine was used to detect and quantify the amount of BrdU incorporation.

Cell growth and survival were measured by BrDU incorporation (Roche) and MTT reduction. HT-29 cells were seeded at a density of 0.5 × 10⁴/well in 96-well microplates and cultured for 24 hours in complete medium, including 10% FCS. After 24 hours of starvation in medium without FCS, purified recombinant endocan (glycanated human endocan, endocan/S137A, glycanated mouse endocan, endocan/S138A) was added at various concentrations ranging from 1 to 1000 ng/mL. After 24 hours of culture, BrDU incorporation and MTT viability assay were performed as recommended by manufacturer. Mitomycine (100 ng/mL) was used as control.

INS-1 β-cells (1.5 × 10⁴ cells/well) and AsPC-1 (5 × 10³ cells/well) pancreatic cancer cells were plated in a 96 well plate and synchronised in low serum media (0.5% FBS) for 24 hrs. Post synchronisation, the cells were either provided with fresh untreated medium or with medium containing various concentrations of rFSTL-1 (R&D Systems) and cultured for further 72 hrs. Cell growth was monitored every 12 hrs during this period using the IncuCyteZOOM live cell imaging system (Essen Bioscience). BrdU incorporation (Roche Applied Science) was measured for the last 24 hrs of the 72 hr culture according to the manufacturer’s instructions. Statistical significance was calculated using Student’s t-test (unpaired, two-tailed) or one-way ANOVA and a p-value < 0.05 was considered significant.

SA-β-Gal staining was performed using a SA-β-Gal staining Kit (9860; Cell signaling Technology) according to the manufacturer’s instructions. Senescent cells were identified as blue-stained cells under light microscopy (AxioVert 135T; Zeiss). BrdU incorporation assays (11647229001; Roche) was performed according to the manufacturer’s guidelines.