n-butanol extract from Folium isatidis inhibits the lipopolysaccharide-induced downregulation of CXCR1 and CXCR2 on human neutrophils

Wu, Beibei; Wang, Liyin; Jiang, Lili; Dong, Lili; Xu, Fengli; Lu, Yili; Jin, Jiahui; Wang, Zhanyue; Liang, Guang; Shan, Xiaoou

Isolation of neutrophils. The present study was approved by the Ethics Committee of The Second Hospital of Wenzhou Medical University (Wenzhou, China), and informed consent was obtained. Peripheral blood was collected from four 20-30-year-old female healthy volunteers between August and December 2015, who had been referred to The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University and who provided informed, written consent. The blood was transferred into heparin lithium-containing tubes (BD Bioscience, San Jose, CA, USA). The samples were processed within 1 h of collection. Whole blood was incubated with 3% dextran T-500 in the dark at room temperature for 20 min for sedimentation of red blood cells (RBCs). Following sedimentation, the white blood cell-enriched upper phase was layered over a Ficoll-Paque solution (GE Healthcare Life Science, Uppsala, Sweden). Centrifugation was performed at 427 x g at room temperature for 20 min without breaks, resulting in four distinct layers. The first three layers were discarded, and the remaining layer containing granulocytes and remnant RBCs was diluted in ACK lysis buffer (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were then washed with PBS, and the freshly isolated neutrophils were resuspended in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.). The purity of the isolated neutrophils was >90% based on Wright's staining and differential counts.