NanoFCM Flare & Auto20 フローナノアナライザー


Richard Zieren, David Clark, Liang Dong, Leandro Ferreira Moreno, Morgan Kuczler, Kengo Horie, Louis Vermeulen, Hui Zhang, Sarah Amend, Theo de Reijke, and Kenneth Pienta
The Journal of Urology, 2021, 206.Supplement 3: e704-e704.



Renal cancer (RCC) accounts for over 73,750 new cases and 14,830 deaths annually in the US. Non-invasive biomarkers are needed to distinguish benign from RCC in small renal masses and to distinguish between clear cell RCC (ccRCC) and papillary RCC (pRCC) subtypes. Extracellular vesicles (EVs), such as exosomes, are a promising source of biomarkers for RCC. Compared with EVs enriched from plasma and urinary, EVs enriched from conditioned cell media (CCM) are readily available and their cellular origin is specific. In this study we aimed to select potential biomarkers by analyzing mRNA and protein cargo of EVs from various RCC and immortalized benign kidney epithelial cell lines and assess biological functions of the EV cargo.


We used the following cell lines; 786O, 769P, and Caki1 (all ccRCC); ACHN, and Caki2 (all pRCC); and HK2, and RPTEC (benign epithelial kidney cells). CCM EVs were enriched by a combination of differential centrifugation, ultrafiltration, and size exclusion chromatography. All CCM EVs were counted by NanoFCM, negative stained and imaged by TEM, and assessed for EV-markers (CD63, CD81, Flot1) by Western blot. The Qiagen miRNeasy kit was used to extract RNA from CCM EVs, the NanoString nCounter low RNA input kit was used for RNA amplification in combination with the Nanostring nCounter PanCancer Progression assay. CCM EVs were processed by Tandem Mass Tag mass spectrometry (TMT MS).


Counts by NanoFCM demonstrated ACHN secreted the most EVs per million cells (1.4×10e7) and Caki-1 the least (5.6×10e4) EVs per million cells. Western blot expression of EV-associated markers CD81, CD63, CD9, and Flot1 was highly correlated with marker expression detected by TMT MS. In mRNA-analyses we found abundance of 74 unique mRNAs in benign EVs, 12 in ccRCC EVs, and 29 in pRCC EVs. By MS analysis we identified 1,726 proteins of which 186 proteins were enriched (> 1.5-fold change) in all types of EVs compared to their parental cell lysates. We found 83-124 proteins with increased abundance in pRCC EVs compared with benign EVs and 83-121 proteins with decreased abundance. In ccRCC EVs we found 95-141 increased abundance compared with benign EVs and 116-130 with decreased abundance.


We compared CCM EVs derived from ccRCC, pRCC, and benign renal cell lines using a multi-omics approach. We used different experimental designs for the mRNA-analysis and mass spectrometry due to technical limitations, yet we were able to analyze exosomal cargos of benign and malign kidney cells. We identified several potential RCC-biomarkers that require further clinical validation.

Source of Funding:

Stichting Cure for Cancer (Amsterdam)


Comparison of the Variability of Small Extracellular Vesicles Derived from Human Liver Cancer Tissues and Cultured from the Tissue Explants Based on a Simple Enrichment Method
Jie Chen, Zhigang Jiao, Jianwen Mo, Defa Huang, Zhengzhe Li, Wenjuan Zhang, Tong Yang, Minghong Zhao, Fangfang Xie, Die Hu, Xiaoxing Wang, Xiaomei Yi, Yu Jiang & Tianyu Zhong.
Stem Cell Reviews and Reports, 18, pages1067–1077 (2022).


A potential use of small extracellular vesicles (sEVs) for diagnostic and therapeutic purposes has recently generated a great interest. sEVs, when purified directly from various tissues with proper procedures, can reflect the physiological and pathological state of the organism. However, the quality of sEV is affected by many factors during isolation, including separation of sEV from cell and tissues debris, the use of enzymes for tissue digestion, and the storage state of tissues. In the present study, we established an assay for the isolation and purification of liver cancer tissues-derived sEVs (tdsEVs) and cultured explants-derived sEVs (cedsEVs) by comparing the quality of sEVs derived from different concentration of digestion enzyme and incubation time. The nano-flow cytometry (NanoFCM) showed that the isolated tdsEVs by our method are purer than those obtained from differential ultracentrifugation. Our study thus establishes a simple and effective approach for isolation of high-quality sEVs that can be used for analysis of their constituents.


Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms
Diogo Fortunato,Danilo Mladenović ,Mattia Criscuoli,Francesca Loria,Kadi-Liis Veiman,Davide Zocco,Kairi Koort and Natasa Zarovni.
International journal of molecular sciences, 2021, 22.19: 10510.


The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

Keywords: extracellular vesicles; exosomes; flow cytometry; nanoparticle tracking analysis; fluorescent dyes; purification; isolation; subpopulations; tetraspanin; antibody


Secreted Ligands of the NK Cell Receptor NKp30: B7-H6 Is in Contrast to BAG6 Only Marginally Released via Extracellular Vesicles
Viviane Ponath,Nathalie Hoffmann,Leonie Bergmann,Christina Mäder,Bilal Alashkar Alhamwe,Christian Preußer and Elke Pogge von Strandmann.
International journal of molecular sciences, 2021, 22.4: 2189.


NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced NKp30 mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions.

Keywords: extracellular ligands; BAG6; B7-H6; NKp30; tumor cell killing


Whole-Transcriptome Analysis of Serum L1CAM Captured Extracellular Vesicles Reveals Neural And Glycosylation Changes In Autism Spectrum Disorder
Yannan Qin, Li Cao, Haiqing Zhang, Shuang Cai, Jinyuan Zhang,Bo Guo,Fei Wu,Lingyu Zhao
,Wen Li,Lei Ni,Liying Liu,Xiaofei Wang,Yanni Chen,Chen Huang.
Journal of Molecular Neuroscience,72, pages1274–1292 (2022)


The pathophysiology of autistic spectrum disorder (ASD) is not fully understood and there are no diagnostic or predictive biomarkers. Extracellular vesicles (EVs) are cell-derived nano-sized vesicles, carrying nucleic acids, proteins, lipids and other bioactive substances. As reported, serum neural cell adhesion molecule L1 (L1CAM)-captured EVs (LCEVs) can provide reliable biomarkers for neurological diseases; however, little is known about the LCEVs in children with ASD. In this study, serum samples were collected from 100 ASD children and 60 age-matched typically developed (TD) children. LCEVs were isolated and characterized meticulously. Whole-transcriptome of LCEVs was analyzed by lncRNA microarray and RNA-Sequencing. All raw data was submitted on GEO Profiles, and GEO accession numbers is GSE186493. RNAs expressed differently in LCEVs from ASD sera vs. TD sera were screened, analyzed, and further validated. A total of 1418 mRNAs, 1745 lncRNAs and 11 miRNAs were differentially expressed, and most of them were down-regulated in ASD. Most RNAs were involved in neuron- and glycan-related networks implicated in ASD. The levels of EDNRA, SLC17A6, HTR3A, OSTC, TMEM165, PC-5p-139289_26, and hsa-miR-193a-5p changed significantly in ASD. In conclusion, whole-transcriptome analysis of serum LCEVs reveals neural and glycosylation changes in ASD, which may help detect predictive biomarkers and molecular mechanisms of ASD, and provide reference for diagnoses and therapeutic management of the disease.


Tissue-derived extracellular vesicles in cancers and non-cancer diseases: Present and future
Su-Ran Li Master, Qi-Wen Man MD, Xin Gao Master, Hao Lin Master, Jing Wang Master, Fu-Chuan Su Master, Han-Qi Wang Master, Lin-Lin Bu MD, Bing Liu MD, Gang Chen MD.
Journal of Extracellular Vesicles, 2021, 10.14: e12175.


Extracellular vesicles (EVs) are lipid-bilayer membrane structures secreted by most cell types. EVs act as messengers via the horizontal transfer of lipids, proteins, and nucleic acids, and influence various pathophysiological processes in both parent and recipient cells. Compared to EVs obtained from body fluids or cell culture supernatants, EVs isolated directly from tissues possess a number of advantages, including tissue specificity, accurate reflection of tissue microenvironment, etc., thus, attention should be paid to tissue-derived EVs (Ti-EVs). Ti-EVs are present in the interstitium of tissues and play pivotal roles in intercellular communication. Moreover, Ti-EVs provide an excellent snapshot of interactions among various cell types with a common histological background. Thus, Ti-EVs may be used to gain insights into the development and progression of diseases. To date, extensive investigations have focused on the role of body fluid-derived EVs or cell culture-derived EVs; however, the number of studies on Ti-EVs remains insufficient. Herein, we summarize the latest advances in Ti-EVs for cancers and non-cancer diseases. We propose the future application of Ti-EVs in basic research and clinical practice. Workflows for Ti-EV isolation and characterization between cancers and non-cancer diseases are reviewed and compared. Moreover, we discuss current issues associated with Ti-EVs and provide potential directions.


Advances in the Field of Micro- and Nanotechnologies Applied to Extracellular Vesicle Research: Take-Home Message from ISEV2021
Silvia Picciolini,Francesca Rodà,Marzia Bedoni and Alice Gualerzi.
Micromachines, 2021, 12.12: 1563.


Extracellular Vesicles (EVs) are naturally secreted nanoparticles with a plethora of functions in the human body and remarkable potential as diagnostic and therapeutic tools. Starting from their discovery, EV nanoscale dimensions have hampered and slowed new discoveries in the field, sometimes generating confusion and controversies among experts. Microtechnological and especially nanotechnological advances have sped up biomedical research dealing with EVs, but efforts are needed to further clarify doubts and knowledge gaps. In the present review, we summarize some of the most interesting data presented in the Annual Meeting of the International Society for Extracellular Vesicles (ISEV), ISEV2021, to stimulate discussion and to share knowledge with experts from all fields of research. Indeed, EV research requires a multidisciplinary knowledge exchange and effort. EVs have demonstrated their importance and significant biological role; still, further technological achievements are crucial to avoid artifacts and misleading conclusions in order to enable outstanding discoveries.

extracellular vesicles; nanomedicine; nanotechnology; theranostics


Comparison of Curative Effect of Human Umbilical Cord-Derived Mesenchymal Stem Cells and Their Small Extracellular Vesicles in Treating Osteoarthritis
Shijie Tang, Penghong Chen, Haoruo Zhang, Haiyan Weng, Zhuoqun Fang, Caixiang Chen, Guohao Peng, Hangqi Gao, Kailun Hu, Jinghua Chen, Liangwan Chen, and Xiaosong Chen.
International Journal of Nanomedicine, 2021, 16: 8185-8202.



Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and their small extracellular vesicles (hUC-MSC-sEVs) have shown attractive prospects applying in regenerative medicine. This study aimed to compare the therapeutic effects of two agents on osteoarthritis (OA) and investigate underlying mechanism using proteomics.


In vitro, the proliferation and migration abilities of chondrocytes treated with hUC-MSCs or hUC-MSC-sEVs were detected by Cell Counting Kit-8 assay and scratch wound assay. In vivo, hUC-MSCs (a single dose of 5 × 105) or hUC-MSC-sEVs (30 μg/time) were injected into the knee joints of anterior cruciate ligament transection-induced OA model. Hematoxylin and eosin, Safranin O/Fast Green staining were used to observe cartilage degeneration. The levels of cartilage matrix metabolic molecules (Collagen II, MMP13 and ADAMTS5) and macrophage polarization markers (CD14, IL-1β, IL-10 and CD206) were assessed by immunohistochemistry. Finally, proteomics analysis was performed to characterize the proteinaceous contents of two agents.


In vitro data showed that hUC-MSC-sEVs were taken up by chondrocytes. A total of 15 μg/mL of sEVs show the greatest proliferative and migratory capacities among all groups. In the animal study, hUC-MSCs and hUC-MSC-sEVs alleviated cartilage damage. This effect was mediated via maintaining cartilage homeostasis, as was confirmed by upregulation of the COL II and downregulation of the MMP13 and ADAMTS5. Moreover, the M1 macrophage markers (CD14) were significantly reduced, while the M2 macrophage markers (CD206 and IL-10) were increased in the hUC-MSCs and hUC-MSC-sEVs relative to the untreated group. Mechanistically, we found that many proteins connected to cartilage repair were more abundant in sEVs. Notably, compared to hUC-MSCs, the upregulated proteins in sEVs were mostly involved in the regulation of immune effector process, extracellular matrix organization, PI3K-AKT signaling pathways, and Rap1 signaling pathway.


Our study indicated that hUC-MSC-sEVs protect cartilage from damage and many cartilage repair-related proteins are probably involved in the restoration process. These data suggest the promising potential of hUC-MSC-sEVs as a therapeutic agent for OA.

Keywords: human umbilical cord-derived mesenchymal stem cells, small extracellular vesicles, osteoarthritis, proteomics, cell-free therapy


Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma
Yang Yang, Yaojie Wang, Sisi Wei, Chaoxi Zhou, Jiarui Yu, Guiying Wang, Wenxi Wang & Lianmei Zhao.
Journal of Translational Medicine, 2021, 19.1: 1-12.



Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application.


In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods.


The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value.


SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.

本研究では超遠心分離法(UC)、サイズ排除クロマトグラフィー(SEC)、市販の分離キット(ExoQuickとexoEasy)の4種類のEVs分離方法を実施し比較検討し、4つの方法で分離したEVのRNAについて、収量、純度、RNAのPCR定量、miRNA-seq解析、mRNA-seq解析の比較を行った。 その結果、SECで得られたEVではmiRNA結合タンパク質AGO2(Argonaute-2)が最も低くなおかつEV特異的miRNAとlncRNAが最も多く観察され、一方で非特異的miRNAの含有量は最も低いことが分かりました。さらにRNA-Seqのデータから、SECで得られたRNAは、miRNAとmRNAの両方について、より有用なリードを提示することが明らかになった。さらに、SECで得られたmRNAは、理想的なFPKM(Fragments Per Kilobase Million)値に匹敵する濃度を持つ傾向があることも判明した。 したがってSECは血漿中のEVを分離するための最適なストラテジーとして、RNomics解析に使用されることが示唆される。

Abnormal expression profile of plasma-derived exosomal microRNAs in patients with treatment-resistant depression
Lian-Di Li, Muhammad Naveed, Zi-Wei Du, Huachen Ding, Kai Gu, Lu-Lu Wei, Ya-Ping Zhou, Fan Meng, Chun Wang, Feng Han, Qi-Gang Zhou & Jing Zhang.
Human genomics, 2021, 15.1: 1-10.


Whether microRNAs (miRNAs) from plasma exosomes might be dysregulated in patients with depression, especially treatment-resistant depression (TRD), remains unclear, based on study of which novel biomarkers and therapeutic targets could be discovered. To this end, a small sample study was performed by isolation of plasma exosomes from patients with TRD diagnosed by Hamilton scale. In this study, 4 peripheral plasma samples from patients with TRD and 4 healthy controls were collected for extraction of plasma exosomes. Exosomal miRNAs were analyzed by miRNA sequencing, followed by image collection, expression difference analysis, target gene GO enrichment analysis, and KEGG pathway enrichment analysis. Compared with the healthy controls, 2 miRNAs in the plasma exosomes of patients with TRD showed significant differences in expression, among which has-miR-335-5p were significantly upregulated and has-miR-1292-3p were significantly downregulated. Go and KEGG analysis showed that dysregulated miRNAs affect postsynaptic density and axonogenesis as well as the signaling pathway of axon formation and cell growths. The identification of these miRNAs and their target genes may provide novel biomarkers for improving diagnosis accuracy and treatment effectiveness of TRD.

治療抵抗性うつ病患者から血漿中EVを分離し、EV中のmiRNAをmiRNA sequencingで解析し、画像収集、発現差解析、標的遺伝子GO濃縮解析、KEGGパスウェイ濃縮解析を行い、miRNAとその標的遺伝子の同定による治療抵抗性うつ病の診断精度や治療効果を向上させるための新規バイオマーカーの推定を行った。


H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans
Fangtao Li,Jiyu Liu,Jizhe Yang,Haoran Sun,Zhimin Jiang,Chenxi Wang,Xin Zhang,Yinghui Yu,Chuankuo Zhao,Juan Pu,Yipeng Sun,Kin-Chow Chang,Jinhua Liu ,Honglei Sun.
PLoS pathogens, 2021, 17.12: e1010098.


H5N6 highly pathogenic avian influenza virus (HPAIV) clade not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

インフルエンザウイルス粒子の組み立てと放出におけるM1タンパク質の役割を調べるため、H5N1由来のeGFP-M1またはH9N2由来のeGFP-M1 VLPsの定量化および、H9N2ウイルス由来のeGFP-M1 EVの定量化をNanoFCMで行った。


Misinterpretation of solid sphere equivalent refractive index measurements and smallest detectable diameters of extracellular vesicles by flow cytometry
van der Pol, Edwin et al. “Misinterpretation of solid sphere equivalent refractive index measurements and smallest detectable diameters of extracellular vesicles by flow cytometry.” Scientific reports vol. 11,1 24151. 17 Dec. 2021, doi:10.1038/s41598-021-03015-2.


Flow cytometers are utilized to characterize single submicrometer particles in biofluids, such as extracellular vesicles (EVs) and viruses. However, because calibration of the optical signals measured by flow cytometers requires optical modelling and valid assumptions, which are not straightforward, statements in the literature about the sensitivity of flow cytometers are often either lacking or incorrect. In this Letter, we explain why measurement artifacts and a statistical artefact cause an overestimation of the refractive index of EVs as derived by Brittain et al.1. Because these artifacts lead to conclusions that are not supported by the data, we re-analyzed the data of Brittain et al. to rectify statements about the sensitivity of their flow cytometer.
The CytoFLEX used by Brittain et al. is a flow cytometer of which the optics have been designed before 20122. The first peer-reviewed scientific publications using the CytoFLEX date from 20163,4. The CytoFLEX houses several optical solutions, such as the catadioptric flow-cell design to maximize light collection, silicon avalanche photodiodes with higher light-detection sensitivity across a larger wavelength range than commonly used photomultiplier tubes, and wavelength-division multiplexing to limit signal loss, which were innovative at the time of introduction. Without any doubt, the optical solutions contribute to achieving single nanoparticle sensitivity, where every photon counts. However, the improvement attributed to the catadioptric flow-cell, as claimed by Brittain et al.1, must be nuanced. Whereas the full-width solid collection angle of the CytoFLEX is 110°, the full-width solid collection angle of other widely used flow cytometers, such as FACSCalibur, FACSCanto, FACSVerse, Gallios, Fortessa, and Navios, is ≥ 104°, but not 30°–60°, as the authors write.
Brittain et al. demonstrate detection of 70 nm polystyrene beads, 99 nm silica beads, and the 95 nm HAdV-5 virus using the violet side scatter detector. The sensitivity of the CytoFLEX is thereby similar to the flow cytometers developed by Hercher et al. in 19795 and Steen in 20046, who measured T2 bacteriophages and 74 nm polystyrene beads by scatter detection, respectively, but (99/24)6 ≈ 4.9 × 103 times less sensitive than the flow cytometer developed by Zhu et al.7, who measured 24 nm silica beads.

  1. Brittain GC, et al. A novel semiconductor-based flow cytometer with enhanced light-scatter sensitivity for the analysis of biological nanoparticles. Sci. Rep. 2019;9:1–13.
  2. Chen, Y. Q. Flow Cytometer. US 10,209,174 B2, 1–69 (2019).
  3. Li T, et al. Immuno-targeting the multifunctional CD38 using nanobody. Sci. Rep. 2016;6:27055. doi: 10.1038/srep27055.
  4. Colombo M, et al. Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies. Nat. Commun. 2016;7:13818. doi: 10.1038/ncomms13818.
  5. Hercher M, Mueller W, Shapiro HM. Detection and discrimination of individual viruses by flow cytometry. J. Histochem. Cytochem. 1979;27:350–352. doi: 10.1177/27.1.374599. 
  6. Steen HB. Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A. 2004;57:94–99. doi: 10.1002/cyto.a.10115.
  7. hu S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano. 2014;8:10998–11006. doi: 10.1021/nn505162u.