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

Flow cytometry. The isolated neutrophils were diluted to 1x10 6 cells/ml with RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.). The cells were then incubated with either vehicle (0.01% DMSO) or increasing concentrations of n-butanol extract from Folium isatidis (100, 250 and 500 µg/ml) at 37˚C and 5% CO2 for 2 h. The specific method to obtain n-butanol extract from F. isatidis was described in previous study (22) . The cells were then stimulated with 0.5 µg/ml lipopolysaccharide (LPS; Sigma; Merck Millipore, Darmstadt, Germany) for 4 h at 37˚C. The cells were washed with ice-cold PBS and then resuspended in 50 µl FACS buffer. The cells were then incubated with a 1:50 dilution of the following antibodies (the concentrations suggested by the manufacturer) for 30 min at 4˚C in the dark: FITC-conjugated anti-human CD181 (CXCR1; cat. no 11-1819-42), PE-conjugated anti-human CD182 (CXCR2; cat. no 12-1829-42) and APC-conjugated anti-human CD11b (cat no. 17-0118-42) or FITC-conjugated anti-human L-selectin (CD62L; cat. no 11-0629-42), PE-conjugated anti-human TLR4 (cat no. 12-9917-41) and APC conjugated anti-human TLR2 (cat no. 17-9922-42) (all from eBioscience, San Diego, CA, USA). Antibodies of the same isotype were used as negative controls. The cells were analyzed using a FACS calibur system (BD Biosciences). The mean fluorescence intensity (MFI) for 10,000 cells in each sample was determined using CellQuest software, version 5.2 (BD Biosciences).