5 bromo 2 deoxyuridine

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.

Cells were incubated with BrdU (5-Bromo-2′-deoxyuridine, 10 µM, Invitrogen) for 3 h, and analyzed after DAPI staining using an ARIA II (BD Biosciences, France). Analysis was performed on 10,000-gated events. Violet laser (405 nm) and Pacific Blue filter were used for DAPI detection, and Red laser (640 nm) and APC filter for BrdU detection. Data analysis and figure generation were performed using the FACS Diva version 6.1.2 program (BD Biosciences, France).

Cardiomyocytes were isolated from the hearts of neonatal Sprague Dawley rats that were ≤3 days old (SLAC Laboratory Animal Co., Ltd., Shanghai, China), and the isolated cells were cultured as described previously with minor modifications (27 (link)). To obtain the cardiomyocytes, heart tissue was digested with 0.1% collagenase type II (Invitrogen Life Technologies) and 0.125% pancreatin (Sigma-Aldrich, St. Louis, MO, USA) for 8 min at 37°C. Following centrifugation, the supernatant was discarded and the cell pellets were resuspended in culture media containing 10% FBS. These steps were repeated until the hearts were completely digested. Cells were pre-plated for 90 min to allow fibroblasts to attach and to yield a purer cardiomyocyte population. The cardiomyocytes were maintained in DMEM containing 10% FBS, 0.1 mM 5-bromo-2′-deoxyuridine (Invitrogen Life Technologies) and 100 IU/ml 0.3% penicillin-streptomycin to inhibit the growth of other cell types. Following 3 days in culture, the cardiomyocytes were subjected to subsequent experimentation.
The cardiomyocytes were divided into the following eight groups: Normoxia (N), TSPG-treated normoxia (N + TSPG), Rg1-treated normoxia (N + Rg1), SM-treated normoxia (N + SM), I/R, TSPG-treated I/R (I/R + TSPG), Rg1-treated I/R (I/R + Rg1) and SM-treated I/R (I/R + SM).

About 100 mg BrdU (5-Bromo-2′-Deoxyuridine) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were dissolved in 32.5 mL anhydrous DMSO (Sigma-Aldrich, Burlington, MA, USA) to prepare a 10 µM stock solution. Then 10 µL of stock solution was diluted in 10 mL of 37 °C tissue culture medium to make a 10 µM labeling solution. The cells were trypsinized, counted, and collected in a sterile tube. Then, the cells were removed from the culture medium, placed in a BrdU labeling solution, and incubated at 37 °C for 2 h. The labeling solution was removed and the cells were washed two times with PBS. Finally, the cells were washed twice with a complete growth medium to be ready for transplantation [18 (link)].