To determine the platelet mitochondrial inner transmembrane potential (ΔΨm), the pre-treated mice platelets were incubated with TMRE to a final concentration of 100 nM. The pre-treated platelet samples were further incubated with TMRE at 37°C in the dark for 20 min and ΔΨm was examined by flow cytometry.
Reactive oxygen species (ROS) were detected in the pre-treated platelets using 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the manufacturer’s instructions (Beyotime, China). Briefly, the pre-treated mouse platelets were further incubated with 10 μM DCFH-DA in the dark at 37°C for 15 min, and the platelets were washed with MTB three times. The platelets were then treated with FCCP (5 μM) or vehicle at RT for 120 min or hypoxia (2% oxygen) for 120 min. The DCF fluorescence intensity of the platelets was examined by flow cytometry.
For detection of P-selectin surface expression, pre-treated mice platelets were further treated with anti-P-selectin antibody or control mouse IgG at RT for 0.5 hr, and then incubated with FITC-GAM at RT in the dark for 0.5 hr. The platelets were then subjected to flow cytometry analysis.
Reactive oxygen species (ROS) were detected in the pre-treated platelets using 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the manufacturer’s instructions (Beyotime, China). Briefly, the pre-treated mouse platelets were further incubated with 10 μM DCFH-DA in the dark at 37°C for 15 min, and the platelets were washed with MTB three times. The platelets were then treated with FCCP (5 μM) or vehicle at RT for 120 min or hypoxia (2% oxygen) for 120 min. The DCF fluorescence intensity of the platelets was examined by flow cytometry.
For detection of P-selectin surface expression, pre-treated mice platelets were further treated with anti-P-selectin antibody or control mouse IgG at RT for 0.5 hr, and then incubated with FITC-GAM at RT in the dark for 0.5 hr. The platelets were then subjected to flow cytometry analysis.