Background:The comparative analysis of ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation for the isolation of exosomes in gouty arthritis synovial fluid (GASF) is rarely reported, and it is not known whether different isolation methods can influence subsequent cytokine analysis.
Methods:GA patients were enrolled during a 1-year period from May 2021 to May 2022. Morphology, particle number, size, purity, protein concentration, and biomarker proteins of GASF-derived exosomes in both extraction methods were observed using transmission electron microscopy, nanoparticle tracer analysis, bicinchoninic acid assay, and Western blotting. An ELISA-based assay platform was used to detect the cytokines in exosomes using Meso Scale Discovery.
Results:Thirty-two cases of fresh GASF were taken and randomly divided between the UC group (n = 16) and the PEG group (n = 16). Transmission electron microscopy images and nanoparticle tracer analysis results showed round vesicles measuring 100 nm on average. The protein expressions of TSG101, CD63, and CD81 in exosomes of the 2 groups were measured via Western blotting. The number and protein concentration of GASF-derived exosome particles from the PEG group were significantly higher than that of the UC group (P < .001). However, in the purity estimation, the UC group reflected significantly higher exosomes extractability (P < .01). Expression of IL-6 and IL-8 in the GASF-derived exosomes were higher in the UC group (P < .05), showing a median of 3.31 (interquartile range, IQR: 0.84-13.16) pg/mL, and a median of 2.87 (IQR: 0.56-13.17) pg/mL, respectively; moreover, IL-1β was mostly undetectable in the PEG group.
Conclusion:The UC method was found to yield exosomes of a higher purity, albeit at a lower quantity but with more abundant inflammatory cytokines; whereas the opposite was the case for the PEG group. The chemical precipitation method might not be suitable in terms of extracting GASF-derived exosomes for inflammation and immunity studies.

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乐备实（上海优宁维生物科技股份有限公司旗下全资子公司），是国内专注于提供高质量蛋白检测以及组学分析服务的实验服务专家，自2018年成立以来，乐备实不断寻求突破，公司的服务技术平台已扩展到单细胞测序、空间多组学、流式检测、超敏电化学发光、Luminex多因子检测、抗体芯片、PCR Array、ELISA、Elispot、PLA蛋白互作、多色免疫组化、DSP空间多组学等30多个，建立起了一套涵盖基因、蛋白、细胞以及组织水平实验的完整检测体系。

 
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