Click it plus edu alexa fluor 488 imaging kit

For a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells were plated in 96-well plates (10⁴ cells in each well). The cell number was determined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. For the EdU incorporation assay, islets were treated with 10 μmol/l EdU (24 h for mouse islets and 48 h for human islets) and stained with anti-insulin antibody (Abcam, Cambridge, MA, USA, ab7842, ×400) and the Click-iT Plus EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher, Waltham, MA, USA). TUNEL staining was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (EMD Millipore, Temecula, CA, USA). The images were taken using a FluoView FV1000-D confocal laser scanning microscope. The proliferating beta cells were measured for 500 or more insulin-positive islet cells per mouse, or 50 or more human islets (containing more than 12,000 beta cells) per donor in each of the groups. For TUNEL staining, at least 10,000 cells per group were analysed.

Cell proliferation within 3D hydrogels (n=4) at days 1, 4 and 7were assessed qualitatively and quantitatively using Click-iT Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific). Cell-laden hydrogels were incubated with 10 μM EdU at 37°C for 24 hours. The samples were then fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 40 min. Click-iT Plus reaction cocktail was added into the samples and incubated for 40 min in the dark. After another rinse with 3% bovine serum albumin (BSA), the samples were stained using Hoechst 33342 (1:2000) at room temperature. CFs cultured on 2D well plates (5000 cells/well, n=4) were taken as control. A confocal microscope (Leica SP5 X MP, Germany) was used to take fluorescence images of the samples. Acquisition parameters were not changed throughout the imaging of each experiment. The amount of EdU positive cells and Hoechst positive cells were analyzed using the ImageJ program, respectively. The value obtained from the EdU positive cells divided by the total number of Hoechst positive cells was determined to be the percentage of cell proliferation.