ABSTRACT
Objective To investigate the effects of fluid shear stress on rolling adhesion of neutrophils on immobilized platelets under flows. Methods Experiments were performed at the parallel plate flow chamber. Platelets were adhered to the functionalized flow chamber bottom which were coated with vWF-A1 first, and then washed with PBS under wall shear stress (WSS) of 1 Pa for different time (0 min, 2.5 min, 7.5 min). A high-speed camera was used to observe and record the rolling adhesion events of neutrophils on immobilized platelets under 50 mPa WSS, and the adhesion parameters such as the number of adhesion events, the tether lifetime of cells and rolling velocity. Results Neutrophils could specifically bind to the immobilized platelets on vWF-A1-coated bottom of the flow chamber. Mechanical stimulation on immobilized platelets had no effects on the tether lifetime of neutrophils on the platelets, but up-regulated the adhesive ratio of neutrophils on the platelets and slowed down the rolling of neutrophils on the platelets. Conclusions Mechanical stimulation on the immobilized platelets will significantly make the circulating neutrophils to be captured easily and promote the rolling adhesion of neutrophils on platelets.
ABSTRACT
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-selectin-induced calcium signaling of neutrophils in flow.
Subject(s)
Female , Humans , Male , Calcium Signaling , Membrane Glycoproteins , Metabolism , Neutrophils , Metabolism , P-Selectin , Metabolism , Stress, Mechanical , Syk Kinase , MetabolismABSTRACT
BACKGROUND: The artificial supported lipid bilayer membrane is the most similar in vitro biomimetic model of the cell membrane. The artificial high-quality lipid bilayer membrane will provide a critical tool for the study of the microstructure of biological membranes, cell signal transduction, biofilm sensors and drug carriers.OBJECTIVE: To explore the optimal preparation conditions of the lipid bilayer membrane with uniform surface and high mobility and its detection indexes.METHODS: By thin film extrusion, liposomes were filtrated 10 times through the filter membrane with the pore diameter of 0.1 and 0.2 μm respectively, with the unfiltered sample as control. The root mean squared roughness of lipid bilayer membrane was detected by atomic force microscopy to explore the optimal pore diameter of the filter membrane for preparation of the lipid bilayer membrane with high uniformity. 160 μL egg-PC (10 g/L) and 5, 10 or 25 μL NBD-PC were mixed separately. The mobility of lipid bilayer membrane was detected by fluorescence recovery after photobleaching (FRAP) to explore the optimal proportion of egg-PC and NBD-PC.RESULTS AND CONCLUSION: The results of atomic force microscopy showed that the lipid bilayer membrane through the 0.1 μm filter membrane had the highest degree of uniformity (P <0.01), with the root mean squared roughness of(0.432±0.181) nm. By using the filter membrane whose pore diameter was greater than 0.1 μm, the surface uniformity of the lipid bilayer membrane was poor and there were more liposome vesicles on the surface. The FRAP results showed that the lipid bilayer membrane through the 0.1 μm filter membrane had a higher degree of fluorescence recovery. When 160 μL egg-PC (10 g/L) and 10 μL NBD-PC were mixed and filtrated through the 0.1 μm filter membrane, the degree of fluorescence recovery of the lipid bilayer membrane reached 90% and the diffusion coefficient was greater than 1 μm2/s which met the standard of high-quality lipid bilayer membrane. These results suggested the optimum ratio of egg-PC to NBD-PC and the optimum filter membrane pore size for preparation of the lipid bilayer membrane by thin film extrusion.