P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface,and mediates intracellular calcium flux,a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue.Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM),the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy.The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow,increasing P-selectin concentration enhanced cellular calcium signaling,and,force triggered,enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin.This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx,and be along the mechano-chemical signal pathway involving the cytoskeleton,moesin and Spleen tyrosine kinase (Syk).These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectininduced calcium signaling of neutrophils in flow.