Fluorescein isothiocyanate fitc anti mouse sca 1

G-CSF was provided by UBI pharma Inc. (Taipei, Taiwan). Fluorescein isothiocyanate (FITC) anti-mouse Sca-1 and phycoerythrin (PE) anti-mouse Flk-1 antibodies were purchased from eBioscience (San Diego, CA, USA). Isotype-identical antibodies were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). The antibodies listed below were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): rabbit anti-eNOS, anti-p-eNOS, mouse anti-VEGF, anti-α-SMA, and anti-β-actin. Rabbit antibodies for STAT3 and p-STAT3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse antibodies for IL-10 were purchased from Serotec (Oxford, UK). Rat anti-CD31 was obtained from BD Pharmingen (San Diego, CA, USA). Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, Histopaque-1077 (density: 1.077 g/mL), and 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Endothelial growth medium (EGM-2 MV) and growth factors for EPC culture were obtained from Lonza (Morristown, NJ, USA).

To investigate the effects of DHI on EPC mobilization to circulating peripheral blood (PB) in response to tissue ischemia, the fluorescence-activated cell sorting (FACS) Caliber flow cytometer (Becton Dickinson, San Jose, CA, USA) was used to assess EPC mobilization. 100 μL of PB was collected from the inner canthus of KKAy mice. PB was incubated with Fluorescein isothiocyanate (FITC) anti-mouse Sca-1 (eBioscience, San Diego, CA, USA), and phycoerythrin (PE) anti-mouse Flk-1 (VEGFR-2, eBioscience) antibodies [32 (link)]. Isotype-identical antibodies (IgGs) were used as negative controls (eBioscience). After 30 min incubation, cells were lysed with red blood cell lysis buffer, washed with phosphate-buffered saline (PBS), and fixed in 1% paraformaldehyde before analysis. Each analysis included 100,000 events. Double positive Sca-1/Flk-1 cell from the mononuclear fraction were considered to be circulating PB EPCs [33 (link)].