Apoptotic cell assay

Apoptosis was assessed by Annexin V-FITC and PI staining followed by analysis with flow cytometry. The methodology followed the procedures as described in the kit. Briefly, the tissue cells were collected by digestion of pancreatic enzyme, which has no EDTA. Then wash cells three times with cold PBS. Resuspended cells in 1 × binding buffer at a concentration of 1×10⁶ cells/mL and transfer 100 μL (1×10⁵ cells) to a 5 mL culture tube. Then added 5 μL of Annexin V-FITC and 10 μL of PI. Gently vortex the tube and incubate for 15 minutes at room temperature in the dark. Later on, 400 μL of 1 × binding buffer was added to each tube. The stained cells were analyzed by flow cytometry as soon as possible (within an hour).

For detection of DNA fragmentation, terminal deoxynu-cleotidyl transferase (TdT) -mediated dUTP nick end labeling (TUNEL) assay was performed as described previously [32]. Briefly, after in vivo I/R procedure, the myocardium of left ventricular was fixed in 10% buffered formalin and subsequently embedded in paraffin to obtain 5μm-thick sections. DeadEnd™ Fluorometric TUNEL system (Promega) was used to stain for apoptotic nuclei. The numbers of TUNEL-positive cells were expressed as a percentage of total cells. TUNEL-positive nuclei were counted by randomly selecting 10 fields of the mid ventricular section. Nuclei were stained by 4’, 6’-diamino-2-phenylen-dolehydrochloride (DAPI) (Invitrogen, USA) and then visualized under fluorescence microscope (Leica DM5000 B, Leica Microsystems). Six specimens were employed for apoptotic index analysis. The apoptotic index was calculated as the ratio of the number of TUNEL-positive neurons to the total number of nuclei.