scholarly journals Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

scholarly journals Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1316
Author(s):  
Andrea Hujacova ◽  
Jan Sirc ◽  
Kristyna Pekarkova ◽  
Tereza Brozova ◽  
Marie Kostelanska ◽  
...  
Keyword(s):  
Cord Blood ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Control Group ◽  
Free Hemoglobin ◽  
Immune Electron Microscopy ◽  
Gestation Age ◽  
Large Platelet ◽  
And Control ◽  
Preterm Newborns

Different biomarkers are investigated to detect the causes of severe complications in preterm infants. Extracellular vesicles (EVs) are recognized as an important part of cell-to-cell communication, and their increased levels were reported in numerous pathological states. We aimed to increase our knowledge about the incidence of platelet and endothelial EVs in cord blood of preterm newborns using conventional flow cytometry. The presence of platelet (CD36+CD41+), activated platelet (CD41+CD62+), and endothelial (CD31+CD105+) EVs was analyzed. Immune electron microscopy was used to confirm the presence of EVs and the specificity of their labeling. The size of detected extracellular vesicles was in the range 400–2000 nm. The differences in the counts of EVs between the preterm and control group were not significant and no correlation of EVs count with gestation age was recorded. Cord blood plasma samples with free hemoglobin level > 1 mg/mL had more than threefold higher counts of CD36+CD41+ and CD41+CD62+ EVs (p < 0.001), while the count of CD31+CD105+ EVs was only moderately increased (p < 0.05). Further studies utilizing cytometers with improved sensitivity are needed to confirm that the analysis of large platelet and endothelial EVs mirrors the quantitative situation of their whole plasma assemblage.

scholarly journals Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

2020 ◽  
Author(s):  
Joana Maia ◽  
Silvia Batista ◽  
Nuno Couto ◽  
Ana C. Gregório ◽  
Cristian Bodo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Population Analysis ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Sample Volume ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Clinical Biomarkers

AbstractExtracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

A Lin-CD45-CD34+ Population of Extracellular Vesicles in Human Blood That Mimics Very Small Embryonic-Like Stem Cells (VSELs) by Flow Cytometry

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4750-4750
Author(s):  
David W. O'Neill ◽  
Yajuan Jiang ◽  
Elizabeth Leary ◽  
Gregory Yavanian ◽  
Sarah Eminli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Extracellular Vesicles ◽  
Umbilical Cord Blood ◽  
Stock Options ◽  
Minor Population ◽  
Two Populations

Abstract Abstract 4750 In mice, Very Small Embryonic-like Stem cells (VSELs) are a population of small (4 to 8 μm) Lin-CD45-Sca1+ bone marrow cells that express CD133, CD34 or CXCR4, as well as markers characteristic of embryonic stem cells such as Oct-4 and Nanog. Molecular as well as in vitro and in vivo functional studies indicate that murine VSELs are pluripotent and capable of regenerating damaged host tissues. Although there is some controversy as to whether the Lin-CD45- population in humans contains cells with stem activity, a sub-population of Lin-CD45- cells with a phenotype very similar to murine VSELs has been identified in human umbilical cord blood as well as in adult human bone marrow and G-CSF mobilized peripheral blood (PB); the latter has shown multi-lineage reconstitution in a mouse bone regeneration model. To isolate these human VSELs for further characterization from G-CSF mobilized adult PB, we used a 4-color panel of antibodies (anti-CD45 Pacific Blue, CD34-PE, CD133-PE, FITC-conjugated antibodies to WBC, RBC and PLT lineage markers, and the cell viability dye 7-AAD) to track the cells during purification. We observed that events with the VSEL phenotype (Live Lin-CD45-CD34/133+ flow cytometry events) are relatively rare, approximately 1 for every 40 CD45midCD34+ hematopoietic progenitor cells. However, using methods that separate cells based on size or density such as differential centrifugation, percoll gradient centrifugation, and counterflow centrifugal elutriation, we observed that these Lin-CD45-CD34/133+ events fall into two separate populations with different physical characteristics – a major population (approximately 98% of Lin-CD45-CD34/133+ events) of objects that are very small (< 4 μm), very light, and that stain negatively or dimly with the nuclear dye DRAQ5, and a minor population that is larger (5–10 μm), heavier, and that stains brightly with DRAQ5. FACS sorting of the two populations followed by cytospin and diff-quick stain showed that the minor population consists of small nucleated cells, whereas the major population consists of membrane-bound objects that do not have a cell nucleus. By light microscopy and transmission electron microscopy these objects have the appearance of extracellular vesicles ranging in size from 0.5 to 3 μm. Although roughly the size of platelets, their morphologic appearance is quite different from platelets. The vesicles contain RNA, which should prove useful in determining the origin and possible function of the vesicles. Initial experiments indicate that the vesicles do not stain for CD31, which would suggest they are not of endothelial origin. The two populations of Lin-CD45-CD34/133+ events are also found in umbilical cord blood, although at different frequencies than in mobilized adult blood (1 for every 5 hematopoietic progenitors, with 94% of the events being DRAQ5-). The data indicate that the addition of a nuclear marker such as DRAQ5 is needed to quantify and track true cells having the VSEL phenotype by flow cytometry. Disclosures: O'Neill: NeoStem, Inc.: Employment, Stock Options Other. Jiang:NeoStem, Inc.: Employment, Stock Options Other. Leary:NeoStem, Inc.: Employment, Stock Options Other. Yavanian:NeoStem, Inc.: Employment, Stock Options Other. Eminli:NeoStem, Inc.: Employment, Stock Options Other. Marasco:NeoStem, Inc.: Equity Ownership.

scholarly journals Antibody-Free Labeling of Malaria-Derived Extracellular Vesicles Using Flow Cytometry

Biomedicines ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 98
Author(s):  
Elya Dekel ◽  
Paula Abou Karam ◽  
Yael Ohana-Daniel ◽  
Mirit Biton ◽  
Neta Regev-Rudzki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Malaria Parasite ◽  
Flow Cytometer ◽  
Cellular Processes ◽  
Parasite Plasmodium ◽  
Membrane Bound ◽  
The Way

Extracellular vesicles (EVs) are cell-derived membrane-bound structures that are believed to play a major role in intercellular communication by allowing cells to exchange proteins and genetic cargo between them. In particular, pathogens, such as the malaria parasite Plasmodium (P.) falciparum, utilize EVs to promote their growth and to alter their host’s response. Thus, better characterization of these secreted organelles will enhance our understanding of the cellular processes that govern EVs’ biology and pathological functions. Here we present a method that utilizes a high-end flow cytometer system to characterize small EVs, i.e., with a diameter less than 200 nm. Using this method, we could evaluate different parasite-derived EV populations according to their distinct cargo by using antibody-free labeling. It further allows to closely monitor a sub-population of vesicles carrying parasitic DNA cargo. This ability paves the way to conducting a more ‘educated’ analysis of the various EV cargo components.

Mer Receptor Tyrosine Kinase Is A Potential Therapeutic Target in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1317-1317
Author(s):  
Alisa B. Lee-Sherick ◽  
Kelly Menachof ◽  
Kristen M. Eisenman ◽  
Amy McGranahan ◽  
Colleen McGary ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Receptor Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Control Cell ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
And Control

Abstract Abstract 1317 Acute myelogenous leukemia (AML) is difficult to treat successfully in both adult and pediatric patients using conventional chemotherapy. Mutated or aberrantly expressed proteins on the cell surface of myeloblasts provide a focus for targeted therapy which could potentially augment therapeutic outcome, decrease toxicity to normal tissues, and/or provide a therapy option for those who are not able to tolerate conventional therapy. We report here that the Mer receptor tyrosine kinase is upregulated in approximately 80% of AML cell lines and patient samples, and explore the therapeutic potential of Mer inhibition. We assessed the prevalence of Mer expression in AML. Western blot and flow cytometric analysis demonstrated expression of Mer in greater than 85% (12/14) of AML cell lines. Mer expression was also assessed at the time of diagnosis and relapse in both pediatric and adult patient samples using flow cytometry. We found that Mer was expressed on leukemic blasts in 80% of 36 pediatric and 100% of 10 adult patients at the time of diagnosis with AML. Additionally, 100% of 11 patients expressed Mer at the time of relapse. Furthermore, when analyzing patient samples at relapse compared to the same patient's diagnostic sample, there was a trend toward increased Mer expression. This is in contrast to normal bone marrow myeloid progenitors from healthy donors, which express little or no Mer. Using two independent shRNA constructs directed against Mer, we analyzed the effects of Mer inhibition in two Mer expressing AML cell lines. Mer knock-down and control cell lines were assessed for apoptosis by flow cytometry after serum starvation and staining with Yo-Pro-1 iodide and propidium iodide. Compared to AML cell lines transduced with a non-silencing control shRNA (shControl), cell lines expressing reduced levels of Mer protein demonstrated significantly more apoptosis (p<0.05). Additionally, when these cell lines were plated in equal number in methylcellulose, cell lines with reduced Mer expression demonstrated decreased colony forming potential compared to shControl cells (p<0.01). Mer knock-down and control cell lines were injected into NOD-SCID-gamma mice after sublethal irradiation and the mice were monitored for development of leukemia. Mice injected with myeloblasts expressing decreased levels of Mer demonstrated significantly prolonged symptom-free survival compared to mice transplanted with shControl AML cells (p<0.001). To further explore the effects of Mer inhibition in AML, we used a novel small molecule tyrosine kinase inhibitor (UNC1666), which has high specificity to Mer. Three Mer expressing AML cell lines were treated with UNC1666 in vitro; treatment reduced phosphorylation of Mer and the downstream signaling molecules ERK1/2 and STAT6. Additionally, treatment with UNC1666 resulted in significant induction of apoptosis (p<0.05) by flow cytometric analysis after staining with Yo-Pro-1 iodide and propidium iodide, and dose-dependent inhibition of colony formation in soft agar, when compared to vehicle treated cells In summary, the upregulation of Mer expression in patient samples and the functional effects on survival with Mer shRNA knockdown help validate Mer as a new and attractive AML therapeutic target. Furthermore, a novel Mer tyrosine kinase inhibitor decreased myeloblast cell survival, providing evidence that Mer is a druggable target in AML. Disclosures: Kireev: WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Wang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

scholarly journals A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
George C. Brittain ◽  
Yong Q. Chen ◽  
Edgar Martinez ◽  
Vera A. Tang ◽  
Tyler M. Renner ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Wavelength Division Multiplexing ◽  
Antigen Expression ◽  
Direct Analysis ◽  
Flow Cytometer ◽  
Light Scatter ◽  
Particle Detection ◽  
Liquid Biopsies ◽  
Double Labeling ◽  
Wavelength Division

Abstract The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Molecular and Circulating Biomarkers of Brain Tumors

2021 ◽  
Vol 22 (13) ◽  
pp. 7039
Author(s):  
Wojciech Jelski ◽  
Barbara Mroczko
Keyword(s):  
Brain Tumors ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Extracellular Vesicles ◽  
Dna Methyltransferase ◽  
Growth Factor Receptor ◽  
Response To Treatment ◽  
Liquid Biopsies ◽  
Methylguanine Dna Methyltransferase ◽  
The Central Nervous System

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations.While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.

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