Anti cd45 pc7

Single platelets and platelet aggregates were enriched from whole blood by density-gradient centrifugation to maximize the efficiency of detecting platelets and platelet aggregates, as described in our previous report [8 (link)]. For analyzing the concentration of platelet aggregates, 500 μl of blood was diluted with 5 ml of saline. Platelets were isolated by using Lymphoprep (STEMCELLS, ST07851), a density-gradient medium, based on the protocol provided by the vendor. Specifically, the diluted blood was layered on the top of Lymphoprep medium and centrifuged at 800 g for 20 min. After the centrifugation, 500 μl of the sample was taken from the mononuclear layer. Platelets were immunofluorescently labeled by adding 10 μl of anti-CD61-PE (Beckman Coulter, IM3605) and 5 μl of anti-CD45-PC7 (Beckman Coulter, IM3548) to the blood sample to ensure the detection of all platelets or platelet aggregates in the sample. Then, 500 μl of 2% paraformaldehyde (Wako, 163–20,145) was added for fixation. Without the fixation, platelet aggregates in the sample would be dismantled. Therefore, the fixation process was performed at least within 4 h after the blood draw. The entire sample preparation was performed at room temperature.