Department of Chemistry, University of Washington, Seattle, Washington, USA.
Micareo Inc, Burlingame, California, USA.
J Extracell Vesicles. 2024 Aug;13(8):e12498. doi: 10.1002/jev2.12498.
High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS/PE particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.
高灵敏度流式细胞仪已被开发用于单外泌体（EVs）的多参数表征，但不同仪器和校准方法的性能存在差异。在这里，我们比较了三种高灵敏度流式细胞仪（两个商业仪器 CytoFLEX/CellStream 和定制的单分子流式细胞仪（SMFC））对源自人结直肠癌细胞（DiFi）的相同（拆分）EV 样本的表征。DiFi EV 用膜染料 di-8-ANEPPS 和 PE 缀合的抗 EGFR 或四跨膜蛋白（CD9/CD63/CD81）抗体染色，用于估计 EV 大小和表面蛋白拷贝数。使用交联硬染色珠进行校准后，免疫荧光和基于粒径的检测限（LOD）为 CytoFLEX 约为 10 PE/约 80nm EV 直径和约 10 PEs/约 67nm CellStream。对于 SMFC，免疫荧光的 LOD 为 1 PE，粒径≤35nm。每个系统检测到的 EV 群体（di-8-ANEPPS/PE 颗粒）差异很大，这取决于系统的 LOD；例如，CellStream/CytoFLEX 分别仅检测到 SMFC 检测到的四跨膜蛋白标记 EV 的 5.7%和 1.5%，并且 CellStream/CytoFLEX 测量和验证的 EV 直径和抗体拷贝数中位数明显大于 SMFC 使用超分辨率/单分子 TIRF 显微镜。为了获得代表所有三个平台分析的常见 EV 群体的数据集，我们根据使用交联硬染色珠进行校准确定的 CytoFLEX LOD，过滤掉 SMFC 和 CellStream 低于 CytoFLEX LOD 的 EV 测量值（10 PE/80nm）。使用此过滤数据集的平台间一致性明显优于未过滤数据集，但通过应用使用 SMFC 数据进行阈值分析确定的更高截止值（21 PE/120nm），可以获得更好的一致性。结果表明，指定 LOD 来定义分析的 EV 群体对 EV 流式细胞术研究中仪器间重现性的影响，以及使用 SMFC 数据进行阈值分析为其他流式细胞仪提供半定量 LOD 值的实用性。
