scholarly journals Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

2020 ◽  
Vol 21 (17) ◽  
pp. 6257
Author(s):  
Donatella Lucchetti ◽  
Alessandra Battaglia ◽  
Claudio Ricciardi-Tenore ◽  
Filomena Colella ◽  
Luigi Perelli ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Binding Sites ◽  
Colorectal Adenocarcinoma ◽  
Adenocarcinoma Cell Line ◽  
Double Positive ◽  
Positive Events ◽  
Light Side ◽  
Sample Dilution ◽  
Monoclonal Antibody Binding ◽  
Intense Research

Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

scholarly journals Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry

2018 ◽  
Vol 64 (4) ◽  
pp. 680-689 ◽  
Author(s):  
Leonie de Rond ◽  
Edwin van der Pol ◽  
Chi M Hau ◽  
Zoltan Varga ◽  
Auguste Sturk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Culture Medium ◽  
Breast Adenocarcinoma ◽  
Analytical Sensitivity ◽  
Adenocarcinoma Cell Line ◽  
Acetoxymethyl Ester ◽  
Fluorescent Markers ◽  
Conditioned Culture Medium ◽  
Carboxyfluorescein Succinimidyl Ester

Abstract BACKGROUND Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry. METHODS Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM+)] or platelet EVs from human plasma [integrin β3 positive (CD61+)]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated. RESULTS Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM+ MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61+ EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection. CONCLUSIONS None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.

1,2-Dihydrochromeno[2,3-c]pyrrol-3-one Derivatives: Synthesis and HPLC-ECD Analysis

Synlett ◽  
2019 ◽  
Vol 30 (07) ◽  
pp. 799-802 ◽  
Author(s):  
László Tóth ◽  
Attila Kiss-Szikszai ◽  
Gábor Vasvári ◽  
Ferenc Fenyvesi ◽  
Miklós Vecsernyés ◽  
...  
Keyword(s):  
Double Bond ◽  
Cell Line ◽  
Antiproliferative Activity ◽  
Reductive Amination ◽  
Colorectal Adenocarcinoma ◽  
Adenocarcinoma Cell Line ◽  
Adenocarcinoma Cell ◽  
Configurational Assignment ◽  
Stereogenic Center

Ethyl-3-formyl-6-methoxy-(2H)-chromene-2-carboxylate was transformed to N-substituted 1,2-dihydrochromeno[2,3-c]pyrrol-3-ones in a domino reductive amination–lactamization reaction. Isomerization of the double bond and the inherently labile stereogenic center was studied, and HPLC-ECD analysis of a chiral 1,2-dihydrochromeno[2,3-c]pyrrol-3(3aH)-one derivative aided by TDDFT-ECD calculation allowed configurational assignment of the separated enantiomers. Antiproliferative activity of the products was demonstrated on the CaCo-2 human epithelial colorectal adenocarcinoma cell line.

Microarray analysis of the differential transformation mediated by Kirsten and Harvey Ras oncogenes in a human colorectal adenocarcinoma cell line

2005 ◽  
Vol 118 (3) ◽  
pp. 616-627 ◽  
Author(s):  
Michael L. Roberts ◽  
Konstantinos G. Drosopoulos ◽  
Ioannis Vasileiou ◽  
Mona Stricker ◽  
Era Taoufik ◽  
...  
Keyword(s):  
Cell Line ◽  
Microarray Analysis ◽  
Colorectal Adenocarcinoma ◽  
Adenocarcinoma Cell Line ◽  
Adenocarcinoma Cell ◽  
Differential Transformation ◽  
Ras Oncogenes

scholarly journals Nanoscale flow cytometry of patient plasma for the detection of prostate cancer-associated extracellular vesicles

2017 ◽  
Vol 3 (2) ◽  
pp. 1-2 ◽  
Author(s):  
Andrew C. Poon ◽  
Johanna Garzon ◽  
Sabine Brett ◽  
Matthew Lowerison ◽  
Karla Williams ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
Benign Prostatic Hyperplasia ◽  
Cancer Patients ◽  
Extracellular Vesicles ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Submicron Particles ◽  
Screening Tests ◽  
Positive Events

Introduction Prostate cancer is the predominant cancer in men, affecting one in seven men during their lifetime. Current tests for prostate cancer include the digital rectal exam and the prostate-specific antigen (PSA) test. Extracellular vesicles (EVs) are submicron particles that participate in intercellular cross-talk by releasing cell mediators such as microRNA, carbohydrates and proteins. While they are known to express the broad tetraspanin family of proteins, i.e. CD9/CD63, prostate cancer-derived EVs have also been found to express PSA and six transmembrane epithelial antigen of the prostate (STEAP1). Traditionally, scientists have purified these EVs through ultracentrifugation. Here we propose a tandem purification of patient plasma, followed by nanoscale flow cytometry (A50+) as a novel method to detect tumour-derived EVs. Materials and Methods Plasma was obtained from healthy volunteers, patients with benign prostatic hyperplasia (BPH), and patients with metastatic prostate cancer. CD9, CD63, PSA and STEAP1 were used as primary antibodies for the purification of EVs from neat plasma. To perform the purification in tandem, Protein G immunoprecipitation using CD9 and PSA was carried out first, followed by immunoaffinity purification with biotinylated CD63 and STEAP1. First and second elutions were collected for the enumeration of dual positive events by A50+. Initial histogram overlays and bivariate plots of neat and purified plasma were computed, then exported as comma-separated values for mathematical modelling by MATLAB. Results Strong dual positive EV populations from patient plasma were optimised, demonstrating that the method enriches tumour-derived EVs from neat samples of patient plasma. This was observed for HVs, patients with benign prostatic hyperplasia, and prostate cancer patients with Gleason 4+4. Enrichment in these purified samples was measured by A50+ and demonstrated by overlaying purified and non-purified histoplots by MATLAB. Additional results show that PSA and STEAP1 can be adapted to detect single or dual positive populations of tumour-associated EVs in prostate cancer patients. Discussions and Conclusions This study suggests that tandem purification of tumour-associated EVs and A50+ analysis from plasma of prostate cancer patients can lead to earlier diagnosis and risk stratification, compared to traditional screening tests and aspiration cytology. Future studies will be directed toward optimizing this detection method for markers of other cancer types to achieve better outcomes in cancer detection and prognosis.

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