PD03-09 ESTABLISHMENT OF A NEW METHOD TO EVALUATE THE PHAGOCYTIC ABILITY OF CALCIUM OXALATE MONOHYDRATE CRYSTALS BY MACROPHAGES

Abstract

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology I1 Apr 2018PD03-09 ESTABLISHMENT OF A NEW METHOD TO EVALUATE THE PHAGOCYTIC ABILITY OF CALCIUM OXALATE MONOHYDRATE CRYSTALS BY MACROPHAGES Atsushi Okada, Hiroshi Takase, Teruaki Sugino, Rei Unno, Kazumi Taguchi, Shuzo Hamamoto, Ryosuke Ando, Keiichi Tozawa, and Takahiro Yasui Atsushi OkadaAtsushi Okada More articles by this author , Hiroshi TakaseHiroshi Takase More articles by this author , Teruaki SuginoTeruaki Sugino More articles by this author , Rei UnnoRei Unno More articles by this author , Kazumi TaguchiKazumi Taguchi More articles by this author , Shuzo HamamotoShuzo Hamamoto More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.275AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It is recently being established that phagocytosis of crystals formed in the kidney by anti-inflammatory macrophages (M2-M&[phis]) inhibits urinary calculi formation, thus preventing stone formation. The aim of this study is to establish a new analytical method for quantifying the phagocytic ability of crystals by macrophages. METHODS Fluorescently labeled calcium oxalate monohydrate (f-COM) crystals were prepared by mixing Alexa Fluor 488 with 200 mM sodium oxalate solution and 200 mM calcium chloride dihydrate solution. After culturing mouse M&[phis]s (J774.1 and RAW264.7) for 24 h, f-COM crystals were exposed at 62.5 μg/cm2 (for 0, 6, and 24 h) and 133 μg/cm2 (for 24 h). At the defined times, the cells were washed with PBS to remove suspended f-COM crystals and then fixed with 4% paraformaldehyde. The cytoplasm of macrophages was stained with Phalloidin-iFluor 555 and the nucleus with 4',6-diamidino-2-phenylindole (DAPI). By using the IN Cell Analyzer 6000, which is an imaging cytometer, 100 fields of view of each well were scanned to quantify the signal intensity of f-COM crystals per cell and the number of phagocytic cells. RESULTS Imaging cytometry revealed f-COM crystals as green and cytoplasm and nucleus of macrophages as red and blue, respectively, in both cell lines. By quantitative analysis, COM signal intensity per cell showed significantly higher RAW 264.7 than J 774.1 at 6 and 24 h. These values also increased significantly with time and COM concentration dependence. Moreover, the COM signal positive cell number showed the same tendency. CONCLUSIONS We developed a method to accurately quantify COM crystals phagocytosed by macrophages. This method is expected to contribute to the development of future drugs that enhance the crystal phagocytic ability of macrophages. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e74-e75 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Atsushi Okada More articles by this author Hiroshi Takase More articles by this author Teruaki Sugino More articles by this author Rei Unno More articles by this author Kazumi Taguchi More articles by this author Shuzo Hamamoto More articles by this author Ryosuke Ando More articles by this author Keiichi Tozawa More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...