Luminal based chemiluminescent probe l 012

Diff-Quik staining solution was purchased from Sysmex (Kobe, Japan). Antibodies against citrullinated histone H3 (citrulline R2 + R8 + R17) and neutrophil elastase were purchased from Abcam (Cambridge, UK). Luminal-based chemiluminescent probe (L-012) and isoflurane were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). The RNeasy® kit was obtained from Qiagen (Hilden, Germany), PrimeScript® 1st strand cDNA Synthesis kit was obtained from Takara Bio (Ohtsu, Japan), and THUNDERBIRD® SYBR qPCR Mix was obtained from Toyobo (Ohtsu, Japan). Novo-Heparin (5000 units), suitable for injection, was purchased from Mochida Pharmaceutical (Tokyo, Japan). RAW264 cells were purchased from Riken BioResource Center (Tsukuba, Japan). ICR mice (6–7 weeks old, male or female) were purchased from Charles River (Yokohama, Japan). The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and were approved by the Animal Care Committee of Musashino University. The sex of the mice did not correlate with the extent of urban aerosol-induced lung injury (number of inflammatory cells and levels of protein in bronchoalveolar lavage fluid (BALF)) (Supplementary Fig. S1). Therefore, male mice were used for all analyses in the present study.