Debnath Sushanta, Nath Bikram, Chakrabarti Abhijit
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI, Kolkata. India.
Protein Pept Lett. 2017;24(10):969-973. doi: 10.2174/0929866524666170818155030.
Misfolding of proteins often leads to aggregation. Accumulation of diverse protein aggregates in various cells, tissue and organs is the hallmark of many diseases, such as Alzheimer's disease and Parkinson's disease.
The main objective of this study was to present a novel method of characterization of protein aggregates, associated with differential toxicity with different size and composition in vitro using flow cytometry.
A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm was used for flow cytometry analysis. The voltage and the gain settings for individual channels were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using Flowing software version 2.5.1 and Origin 8.0.
Autofluorescence and scattering intensities could distinguish between amyloid and nonamyloid aggregates. Dot plots of both side scattering (SSC) and forward scattering (FSC) intensities also showed characteristic fingerprint of both the types of aggregates when compared with those of well known nanoparticles of oxides of Fe and Cu.
This work reports a novel, simple and robust flow cytometric method of characterization of protein aggregates of different size and composition which would find wider application in characterization of biomolecular aggregates, in general.
蛋白质错误折叠常导致聚集。多种蛋白质聚集体在各种细胞、组织和器官中的积累是许多疾病的标志，如阿尔茨海默病和帕金森病。
本研究的主要目的是提出一种新的方法，用于表征蛋白质聚集体，该方法利用流式细胞术在体外对不同大小和组成的具有不同毒性的蛋白质聚集体进行表征。
使用配备488nm氩离子激光器的贝克曼库尔特Epics XL流式细胞仪进行流式细胞术分析。各个通道的电压和增益设置为高电压和高增益，用于检测自发荧光、吸附刚果红的荧光、蛋白质和纳米颗粒聚集体的前向散射（FSC）和侧向散射（SSC）强度。对每个样品进行分析，以表征和量化聚集体数量，最大检测事件数为20,000。使用Flowing软件2.5.1版和Origin 8.0对流式细胞术数据进行分析。
自发荧光和散射强度可区分淀粉样聚集体和非淀粉样聚集体。与已知的铁和铜氧化物纳米颗粒相比，侧向散射（SSC）和前向散射（FSC）强度的点图也显示了两种聚集体类型的特征指纹。
本研究报告了一种新颖、简单且可靠的流式细胞术方法，用于表征不同大小和组成的蛋白质聚集体，总体而言，该方法将在生物分子聚集体的表征中得到更广泛的应用。
