D by two-dimensional targeted M-mode, and the left ventricular ejection fraction (LVEF) was calculated:LVEF??? left ventricular enddiastolic volume 3-Bromo-5-chloro-2-fluoroaniline left ventricular endsystolic volume?=left ventricular enddiastolic volume??The tube formation test is a well-established assay used to detect the formation of three-dimensional vessels and used widely to assess angiogenesis in vitro. Matrigel (Sigma) was prepared in 24-well plate according to the manufacturer's instructions. DMOG-preconditioned (DMOG-BMSCs) or nonpreconditioned BMSCs were seeded into the coated wells at PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 a density of 50,000 cells, and were incubated for 6 hours at 37 for full development of vessel-like tube structures. Tube formation was quantified by the cumulative tube length. In the case where several tube-like structures merged together or branched, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/21715270 the total length of tubes was calculated as the sum of the length of the individual branches.Immunofluorescence stainingUsing echocardiographic evaluation, hemodynamic measurement was performed. After right carotid artery exposure, a microtip catheter was cannulated into the left ventricle and was connected with a MLT0699 disposable pressure transducer (AD Instrument, Colorado Springs, CO, USA). The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum change rate of left ventricular pressure rise and fall ( p/dt), time constant of the isovolumic pressure decline (Tau) and heart rate were monitored and recorded using the Powerlab/800 data acquisition system (AD Instrument).Measurement of infarct sizeFor immunofluorescence staining, heart slices were fixed with 10 formalin for 10 minutes, followed by permeabilization with 0.2 Triton X-100 for 5 minutes and blocked with 1 fish gelatin (Sigma) for 1 hour ijms17122034 at room temperature. Specific primary antibodies were incubated overnight at 4 in a humidified environment, and washed three times with PBS. Specimens were then incubated with Cy3-conjugated Donkey anti-rabbit IgG (1:500; Jackson ImmunoResearsh, West Grove, PA, USA) or Alexa Fluor 488 anti-goat IgG (1:200; Molecular Probes, Carlsbad, CA, USA) for 1 hour at room temperature. Nuclear stainingRats were sacrificed after measuring hemodynamics by an overdose of anesthetic. Hearts were harvested quickly and split into transverse sections: apex, mid left ventricle, and base. The tree sections of the hearts were embedded in optimal cutting temperature compound (Sakura Finetek USA Inc., Torrance, CA, USA). Samples cut at 10 m thickness were stained with Masson's trichrome and images of each slide were digitized through the NIH image analysis system. The percentage of infarct size/fibrotic area was calculated by dividing the sum of epicardial and endocardial circumference of the infarcted area by the sum of the total endocardial and epicardial circumference of the left ventricle [42].Statistical analysisData were analyzed with SPSS 13.0 (IBM Corp. Armonk, NY, USA) and expressed as mean ?standard error of mean. Student's two-tailed tert-Butyl (7-bromoheptyl)carbamate t test was applied forLiu et al. Stem Cell Research Therapy 2014, 5:111 http://stemcellres.com/content/5/5/Page 5 ofcomparison of two independent experimental groups and one-way analysis of variance followed by the Tukey post hoc test for multiple comparisons. Statistical significance was defined as P <0.05.ResultsUpregulation of survival and angiogenic factors in DMOG-preconditioned BMSCsTo evaluate the effect of DMOG preconditioning on BMSCs, we first analyz.