AB0050\u2005A NOVEL METHOD FOR ISOLATION OF EXOSOMES FROM SYNOVIAL FLUID

Abstract

Exosomes in synovial fluid (SF) has a close relationship with the pathogenesis of rheumatiod arthritis. As a complex biological fluid, SF presents challenges for exosomes isolation using standard methods, such as ExoquickTM kit and ultracentrifugation.The study aims to compared the quality of exosomes separated by ExoquickTM kit (TM), ExoquickTM kit+ExoquickTC kit (TM-TC), ultracentrifugation (UC) and TM-TC+UC(TM-TC-UC) from SF.Exosomes was separated by TM, TM-TC, UC and TM-TC-UC respectively. The size and concentrations of exosomes were detected by high sensitivity flow cytometry for nanoparticle analysis. Total protein and RNA were extracted from exosomes. SDS-PAGE was used to detect the protein distribution of exosomes. Western blot was used to examine the level of albumin and exosomes marker (TSG101 and CD81).There was no statistic difference in the diameters of exosomes separated by the four methods. The concentrations of exosomes in TM, TM-TC, TM-TC-UC and UC were (5.65±0.93), (3.02±1.19), (1.67±0.25) and (4.61±0.73) *109Particles/mL. The protein concentrations of exosomes separated by the four methods were consistent with the concentrations of exosomes. SDS-PAGE showed that the protein distribution of exosomes separated by the four methods were different. Low levels of albumin were detected in TM-TC and TM-TC-UC, while high levels of albumin in TM and UC. Total RNA concentrations from exosomes in TM-TC was higher than other groups.TM-TC can be used to obtain higher quality exosomes from SF for the study of exosome-enriched components.[1]Helwa I, et al, A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PloS one, 2017. 12(1): p. e0170628-e0170628.Figure 1.A: SDS-PAGE showed the protein distribution of exosomes; B: the detection of albumin, TSG101 and CD81 by western blot.None declared