Fitc conjugated cd34

Six days after stroke induction, 1 ml of peripheral blood was collected from each mouse by aspiration from the left heart ventricle. Mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (GE Healthcare Biosciences, PA, USA), purified, resuspended in phosphate buffered saline containing 0.5% bovine serum albumin, and then incubated with the following antibodies: FITC-conjugated CD34, PE-conjugated VEGFR2/Flk1/KDR (BD Biosciences, San Jose, CA, USA). Labeled cell populations were measured by FACSCalibur (BD Biosciences). FACS (Fluorescence Activated Cell Sorting) data was analyzed by FlowJo 7.0 software (Ashland, OR, USA). FACS analysis was performed using a variety of controls including unstained samples, isotype antibodies, and single-stained samples for determining appropriate gates, voltages, and compensations required in multivariate flow cytometry.

Cell sorting was performed on the FACSAria (BD Bioscience), and phenotypic analysis was performed on the FACS Canto II (BD Bioscience) using CellQuest Pro Software (BD bioscience and FlowJo software. For cell-surface staining, collected cells were washed twice with ice-cold PBS followed by incubation with saturating concentrations of the appropriate mAbs for 15 min at 4°C and then, were washed twice in ice-cold PBS. For intracellular staining, cells were fixed and rendered permeable using the Fix and Perm kit (BD biosciences), according to the manufacturer's instructions. The antibodies used in this study were FITC-conjugated CD34, CD3 and NKp46, PE-conjugated CD56, NKG2D, NKp30, CD3, CD117, CD132, CD107a and p-STAT5, APC-conjugated CD56, CD94, and APC-Cy7 conjugated CD56 (BD bioscience).