Matching isotype antibodies

For analysis of BMCs within the hearts, a ‘myocyte-depleted’ cardiac cell population was prepared, incubating minced myocardium in 0.1% collagenase IV (GIBCO BrL, Carlsbad, CA, USA) 30 min. at 37°C, lethal to most adult mouse CMs. Cells were then filtered through a 70 mm mesh. To exclude spurious effects of enzymatic digestion, BM cells with or without collagenase treatment were stained revealing no significantly changed staining of labelled cell antigens. Mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (Sigma Chemicals, St. Louis, MO, USA), purified, and resuspended in PBS containing 1% BSA. Cells were incubated for 40 min. in the dark at 4°C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE) and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, VLA-4-PE, c-kit-PE and CXCR4-PE (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen, San Jose, CA, USA) served as controls. Cells were analysed by three-colour flow cytometry using a Coulter® Epics®XL-MCLTM flow cytometer (Beckman Coulter, Cincinnati, OH, USA). Each analysis included 50.000 events.

Ten- to 12-week-old CXCR4-EGFP BAC transgenic reporter mice with or without LAD ligation were either treated with saline or DMOG (80 mg/kg/day) for up to 7 days. BM mononuclear and myocyte-depleted cardiac cells were separated as previously described [4 (link)]. Cells were incubated for 40 min in the dark at 4 °C with the following fluoresceinisothiocyanate (FITC)-, phycoerythrin (PE)-, and peridininchlorophyll-protein (PerCP)-conjugated monoclonal antibodies: CD45-PerCP, CD11b-PErCP, CD11b-PE, CD4-PE, CD20-PE, CD31-PE, CD34-PE, Flk-PE, CD86-PE, CD206-PE, F4/80-PE, CD133-PE, c-kit-PE, Sca-1-PE, CD3-biotin, CD45R/B220-biotin, CD11b-biotin, TER-119-biotin, and Ly-6G-biotin (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen) served as controls. Cells were analyzed by three-color flow cytometer using a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter). Each analysis included 50,000 events.