Pathway 855 imaging system

Seventy-two hours after siRNA transfection, cells were washed three times with PBS, fixed with 4 % (v/v) paraformaldehyde containing 0.02 % (v/v) Triton X-100 (AppliChem) at room temperature for 30 minutes and subsequently stained for 1 h with 1.25 μg/ml of Hoechst, to visualize DNA. Peak intensity, total peak intensity and number of Hoechst stained objects were measured on an Acumen Explorer microplate cytometer ex3 HCS (TTP LabTech), using the following parameters: Hoechst voltage, 550; sliding window, x = 1 μm y = 1 μm. In addition, a population manager was applied setting a minimal and maximum object size to exclude false positive objects. For MSC morphology studies, MSCs were blocked with 1 % bovine serum albumin and additionally stained for 1 h with Alexa Fluor 547 Phalloidin (1:3,000; Life Technologies) and fluorescein isothiocyanate (FITC)-conjugated α-tubulin (1:1000; Sigma). Images were taken using the BD Biosciences Pathway 855 imaging system. Three images were taken from each well at 10× magnification to cover approximately 60 % of the total well area. Images were analyzed using ImageJ (v.1.440). For each experiment at least one positive (UBC) and one negative control (pGL3) were used.

To assess β-cell proliferation in vivo, mice were treated with 0.8 mg/ml of the synthetic nucleoside and thymidine analog 5-bromo-2′-deoxyuridine (BrdU; Thermo Fisher Scientific) 1 wk during the second month of diet treatment. After isolation and sectioning, pancreases were stained for BrdU and insulin to determine the amount of proliferating β cells. The images obtained with the Pathway 855 imaging system (BD Biosciences, San Jose, CA, USA) were analyzed with the Cell Profiler (http://cellprofiler.org/). Nuclei were identified by DAPI staining; the intensities of the insulin and BrdU staining were measured in the nucleus and displayed as histogram. Insulin-positive objects were defined as β cells. BrdU-positive and -negative cells were identified and used for calculating the percentage of BrdU-positive β cells.