CXCR4 and CXCR7 are ligand receptors for SDF-1 and have a role in regulating the biological activities of EPCs. Therefore, the expression levels of CXCR4 and CXCR7 were assessed in each group.
Confocal immunofluorescence microscopy was performed to determine the expression levels of CXCR4 and CXCR7 in EPCs. Briefly, EPCs (1×105 cells/well) were grown on glass coverslips for 12 h, fixed with 4% paraformaldehyde at room temperature for 30 min and incubated with 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min and 1% BSA (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Subsequently, EPCs were incubated with anti-human CXCR4 (1:1,000; Abcam; ab197203) and anti-human CXCR7 monoclonal antibodies (1:1,000; Abcam; ab72100) overnight at 4°C, and then incubated with Alexa Fluor 488-(1:500; Abcam; ab150077) and Alexa Fluor 647-conjugated secondary antibodies (1:500; Abcam; ab150079) for 2 h at room temperature. Cell nuclei were stained with DAPI for 10 min at room temperature. Confocal immunofluorescence microscopy images were captured using a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH).
Confocal immunofluorescence microscopy was performed to determine the expression levels of CXCR4 and CXCR7 in EPCs. Briefly, EPCs (1×105 cells/well) were grown on glass coverslips for 12 h, fixed with 4% paraformaldehyde at room temperature for 30 min and incubated with 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min and 1% BSA (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Subsequently, EPCs were incubated with anti-human CXCR4 (1:1,000; Abcam; ab197203) and anti-human CXCR7 monoclonal antibodies (1:1,000; Abcam; ab72100) overnight at 4°C, and then incubated with Alexa Fluor 488-(1:500; Abcam; ab150077) and Alexa Fluor 647-conjugated secondary antibodies (1:500; Abcam; ab150079) for 2 h at room temperature. Cell nuclei were stained with DAPI for 10 min at room temperature. Confocal immunofluorescence microscopy images were captured using a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH).