Flow cytometry is commonly used to characterize nanoparticles (NPs) and extracellular vesicles (EVs) but results are often expressed in arbitrary units to indicate fluorescence intensity. This hampers interlaboratory and inter-platform comparisons. We investigated the use of molecules of equivalent soluble fluorophores (MESF)-beads for assignment of fluorescence values to NPs and EVs by comparing two FITC-MESF bead sets as calibrators on different flow cytometry platforms (BD Influx, CytoFLEX LX and SORP BD FACSCelesta). Next, fluorescence signals of NPs and EVs were calibrated using different sets of FITC and PE-MESF beads. Fluorescence calibration using beads designed for cellular flow cytometry allowed inter-platform comparison. However, the intrinsic uncertainty in the fluorescence assignment to these MESF beads impacts the reliable assignment of MESF values to NPs and EVs based on extrapolation into the dim fluorescence range. Our findings demonstrate that the use of the same set of calibration materials (vendor and lot number) and the same number of calibration points, greatly improves robust interlaboratory and inter-platform comparison of fluorescent submicron sized particles.

中文翻译：

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荧光校准器的内在变异性影响通过流式细胞术将 MESF 或 ERF 值分配给纳米颗粒和细胞外囊泡

流式细胞术通常用于表征纳米粒子 (NP) 和细胞外囊泡 (EV)，但结果通常以任意单位表示以指示荧光强度。这阻碍了实验室间和平台间的比较。我们通过比较两个 FITC-MESF 珠组作为不同流式细胞术平台（BD Influx、CytoFLEX LX 和 SORP BD FACSCelesta）上的校准器，研究了等效可溶性荧光团分子 (MESF)-珠子将荧光值分配给 NP 和 EV 的用途. 接下来，使用不同组的 FITC 和 PE-MESF 珠校准 NP 和 EV 的荧光信号。使用专为细胞流式细胞术设计的珠子进行荧光校准，可以进行平台间比较。然而，这些 MESF 珠子的荧光分配的内在不确定性会影响基于外推到暗淡荧光范围的 MESF 值到 NP 和 EV 的可靠分配。我们的研究结果表明，使用同一组校准材料（供应商和批号）和相同数量的校准点，大大提高了荧光亚微米尺寸颗粒的稳健实验室间和平台间比较。