815

Abstract

Introduction: Adenosine triphosphate (ATP), which is well known as energy currency of cells, is involved in both intracellular and extracellular signaling. Neutrophils (PMN) have a pivotal role in inflammation response. We have shown that ATP release and feedback via ATP receptors are essential for neutrophil activation. Meanwhile, two new fluorescent chemosensors (2-2Zn(II), 3-2Zn(II)), which detect ATP and its delivertives in specific region of living cells, were developed. Although the utility of these chemosensors has been demonstrated in cultured cells, imaging studies using human PMN have not been reported. Our objective is to establish the methods of staining human PMN with 2-2Zn(II), 3-2Zn(II) and quantify the fluorescence intensity by flow cytometry. Methods: PMN were isolated from the peripheral blood of healthy volunteers (n=12). Live PMN with these staining were observed by a confocal microscopy. Fluorescence intensity on plasma membrane (2-2Zn(II)) and in mitochondria (3-2Zn(II)) of PMN with and without fMLP stimulation were evaluated using flow cytometry. Results: These chemosensors could localize on plasma membrane surface and mitochondria in human neutrophils. Mean fluorescence intensity (MFI) of 2-2Zn(II) on plasma membrane of PMN with fMLP stimulation was significantly higher than without stimulation (274.3 ± 100.6 vs 208.4 ± 81.8 ; p< 0.01). On the other hand, MFI of 3-2Zn(II) in mitochondria with stimulation tends to be lower than without stimulation . Conclusions: Fluorescence imaging of ATP in human PMN with chemosensors (2-2Zn(II), 3-2Zn(II)) was established. The higher MFI on plasma membrane with fMLP stimulation suggests the activation of PMN. This method would contribute to understanding the dynamics of ATP in neutrophils and elucidating their diverse physiological functions in inflammation.