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Analysis of Calcium Transients During Neutrophil Migration by New Dynamic Visualization System TAXIScan
Chen Ting, Liang Haoyue, Geng Guangfeng, Yu Wenying, Yang Wanzhu, Cheng Xuelian, Bai Yang, Fu Weichao, Ren Yiran, Xu Yuanfu*
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
Abstract: To observe and analyze the cytosolic free calcium changes in neutrophils during fMLP (N-formyl-methionyl-leucyl-phenylalanine)-induced cell migration， neutrophils were isolated from bone marrow by centrifugation on discontinuous percoll gradient， and then were stained with Fluo-3/AM. Cytosolic free calcium changes in neutrophils stimulated with different concentrations of fMLP (0.02~100 μmol/L) were detected by flow cytometry. fMLP-induced polarity and cytosolic free calciumions changes in neutrophils during migration were recorded by TAXIScan， and relevant parameters were analyzed by Diva and Volocity softwares. Flow cytometry results showed that maximum peaks of the transient calcium ions were achieved by stimulation of neutrophils with fMLP at concentrations of 0.5， 1 and 5 μmol/L. TAXIScan results showed that fMLP (0.1~10 μmol/L)-induced intracellular calcium transients of neutrophils were observed preferentially， which was followed by polarization and directional migration. And neutrophils migrated with a fastest velocity when stimulated with fMLP (1 μmol/L).These results suggested that fMLP concentration-dependent changes of [Ca2+]i (cytosolic free Ca2+ concentration) could be accurately detected by combined with flow cytometry， and TAXIScan dynamic visualization system. This study also provided a new real-time observation method for analyzing the relationship between polarity and calcium ions in neutrophils in the future.
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