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

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was isolated from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR analysis was performed using an M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 1 µg RNA was used for the determination. qPCR was performed in a 10 µl reaction volume containing 2 µl cDNA target, 2.25 µl SYBR Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 0.25 µl sense primers, 0.25 µl anti-sense primers and 5.25 µl ddH 2 O. An Eppendorf Mastercycler realplex detection system (Eppendorf, Hamburg, Germany) was used for RT-qPCR analysis: The reaction consisted of the following conditions: 95˚C for 3 min, followed by 95˚C for 15 sec, 60˚C for 30 sec, and 70˚C for 30 sec (40 cycles). The primers of the genes were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and the sequences were as follows: Human CXCR1, The result of real-time PCR was expressed as the threshold cycle (CT). The CT represents the PCR cycle at which the reported fluorescence rises above a set baseline threshold when the DNA amplicon is replicating exponentially (12) . Results were normalized to β-actin as plotted as relative expression to the average of CON or DMSO, which were set as 100.