Intrinsic variability of fluorescence calibrators impacts the assignment of MESF or ERF values to nanoparticles and extracellular vesicles by flow cytometry

IF 4.2 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Nanomedicine : nanotechnology, biology, and medicine Pub Date : 2023-11-23 DOI:10.1016/j.nano.2023.102720

Estefanía Lozano-Andrés PhD , Tina Van Den Broeck PhD , Lili Wang PhD , Majid Mehrpouyan PhD , Ye Tian PhD , Xiaomei Yan PhD , Ger J.A. Arkesteijn PhD , Marca H.M. Wauben PhD

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Abstract

Flow cytometry allows to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units of fluorescence. We evaluated the precision and accuracy of molecules of equivalent soluble fluorophores (MESF) beads for calibration of NPs and EVs. Firstly, two FITC-MESF bead sets, 2 and 6 um in size, were measured on three flow cytometers. We showed that arbitrary units could not be compared between instruments but after calibration, comparable FITC MESF units were achieved. However, the two calibration bead sets displayed varying slopes that were consistent across platforms.

Further investigation revealed that the intrinsic uncertainty related to the MESF beads impacts the robust assignment of values to NPs and EVs based on extrapolation into the dim fluorescence range. Similar variations were found with PE MESF calibration.

Therefore, the same calibration materials and numbers of calibration points should be used for reliable comparison of submicron sized particles.

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荧光校准器的内在可变性影响了流式细胞术对纳米颗粒和细胞外囊泡的MESF或ERF值的分配。

流式细胞术可以表征纳米颗粒(NPs)和细胞外囊泡(ev)，但结果通常以任意单位的荧光表达。我们评估了等效可溶性荧光团(MESF)微球分子的精度和准确性，用于NPs和ev的校准。首先，在三台流式细胞仪上测量两个FITC-MESF头组，大小分别为2 um和6 um。我们发现任意单位不能在仪器之间进行比较，但在校准后，可以获得可比较的FITC MESF单位。然而，两个校准头组显示不同的斜度，在各个平台上是一致的。进一步的研究表明，与MESF珠相关的固有不确定性影响了基于外推到微弱荧光范围的np和ev值的稳健分配。在PE MESF校准中也发现了类似的变化。因此，为了对亚微米级颗粒进行可靠的比较，应使用相同的校准材料和校准点的数量。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Nanomedicine : nanotechnology, biology, and medicine 工程技术-纳米科技

CiteScore

11.10

自引率

0.00%

发文量

133

审稿时长

42 days

期刊介绍： The mission of Nanomedicine: Nanotechnology, Biology, and Medicine (Nanomedicine: NBM) is to promote the emerging interdisciplinary field of nanomedicine. Nanomedicine: NBM is an international, peer-reviewed journal presenting novel, significant, and interdisciplinary theoretical and experimental results related to nanoscience and nanotechnology in the life and health sciences. Content includes basic, translational, and clinical research addressing diagnosis, treatment, monitoring, prediction, and prevention of diseases.