C kit pe

The decidual unsorted cells (dUCs; including dCD34⁺ and dCD34^‐ cells) were collected and labelled with the following antibodies for dual staining: CD34‐FITC (BD, Franklin Lakes, NJ, USA)/ c‐kit‐PE (BD) for 30 minutes at 4°C. The dCD34⁺ cells were stained with CD34‐FITC (BD), c‐kit‐PE (BD), CD90‐FITC (BioLegend, San Diego, CA, USA), CD105‐APC (BioLegend), CD31‐FITC (BD), VEGFR‐2‐FITC (BD), VE‐cadherin‐FITC (BD), HLA‐ABC‐FITC (Abcam) and HLA‐DR‐FITC (Abcam). Then, the cells were washed three times with cold PBS before being centrifuged at 1000 rpm for 5 minutes. Immunoreactivity of the cell surface antibody markers was assayed by fluorescence‐activated cell sorting (FACS; BD).

Mice were sacrificed by exsanguination via cardiac puncture and lungs were perfused clear of blood with saline and removed from the thoracic cavity, as previously described [25] . Lung tissue-associated cells were extracted from the right lung by mincing and enzymatic digestion, as previously described [20] (link). Mononuclear cells were collected following density gradient centrifugation (400 g for 20 min) over Histopaque (Sigma, Oakville, Ontario, Canada).
Cells were immunostained with Sca-1-FITC, c-kit-PE (BD Bioscience, Oakville, ON, Canada) and VEGFR2-APC (eBioscience Inc., San Diego, CA, USA), or isotype control antibodies (40 min at 4°C), and fixed in PBS with 1% paraformaldehyde (BDH Laboratory Supplies, Mississauga, ON, Canada), and cell data (100,000 events in the lymphomononuclear region) were acquired using a FACSCaliber flow cytometer equipped with a 488-nm argon ion laser (BD Instrument Systems, Mississauga, ON, Canada). Primitive progenitor cells (Sca-1⁺c-kit⁺) and lineage-committed EPCs (Sca-1⁺c-kit⁺VEGFR2⁺) were enumerated using the Cellquest software package (BD Biosciences). The flow cytometric gating strategy are previously described in detail in [15] (link). Doyle et al., 2011 [20] (link). Absolute numbers of cells were calculated using the percentage of population positivity obtained by flow cytometery and the total white cell count.

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.