scholarly journals BIOM-25. IDENTIFYING EXTRACELLULAR VESICLES FROM GLIOBLASTOMA OR NON-NEOPLASTIC CELLS VIA IMAGING FLOW CYTOMETRY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii7-ii7
Author(s):  
Luz Milbeth Cumba Garcia ◽  
Abudumijiti Aibaidula ◽  
Nazanin Yeganeh Kazemi ◽  
Miyeon Jung ◽  
Fabrice Lucien-Matteoni ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Protoporphyrin Ix ◽  
Molecular Profile ◽  
Cell Of Origin ◽  
Double Positive ◽  
Neoplastic Cells ◽  
Monocytic Cell ◽  
Imaging Flow Cytometry

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.

scholarly journals A method to study extracellular vesicles secreted in vitro by cultured cells with minimum sample processing and extracellular vesicle loss

2021 ◽  
Author(s):  
Anissa Viveiros ◽  
Vaibhavi P Kadam ◽  
John Monyror ◽  
Luis Carlos Morales ◽  
Desmond Pink ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Conditioned Medium ◽  
Extracellular Vesicles ◽  
Cell Biology ◽  
Cultured Cells ◽  
Fluorescent Labeling ◽  
Size Exclusion ◽  
Sample Processing ◽  
Imaging Flow Cytometry

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The study of EV secretion and EV characterization remains challenging due to the small size of these particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV separation techniques. We developed a method for in-cell EV labeling with fluorescent lipids (DiI), followed by DiI-labelled EV characterization in the conditioned medium by imaging flow cytometry (IFC). Direct IFC analysis of EVs in the conditioned medium, after removal of apoptotic bodies and cellular debris, significantly reduces sample processing and loss compared to established methods for EV separation, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by ultracentrifugation. In conclusion, our optimized protocol for EV labeling and analysis reduces EV sample processing and loss, and is well suited for cell biology studies that focus on modulation of EV secretion by cells in culture. Keywords: extracellular vesicles, fluorescent labeling, imaging flow cytometry, ultracentrifugation, size exclusion chromatography, nanoparticle tracking analysis

scholarly journals Conventional, High-Resolution and Imaging Flow Cytometry: Benchmarking Performance in Characterisation of Extracellular Vesicles

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 124
Author(s):  
Jaco Botha ◽  
Haley R. Pugsley ◽  
Aase Handberg
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Extracellular Vesicles ◽  
Single Particles ◽  
Multiple Parameters ◽  
Imaging Flow Cytometry ◽  
Highly Sensitive ◽  
Individual Project ◽  
Benchmarking Performance ◽  
High Throughput Manner

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

scholarly journals Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers

2021 ◽  
Author(s):  
Rachel R. Mizenko ◽  
Terza Brostoff ◽  
Tatu Rojalin ◽  
Hanna J. Koster ◽  
Hila S. Swindell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Fluorescence Detection ◽  
Single Particle ◽  
Cancer Biomarkers ◽  
Surface Markers ◽  
Isolation Method ◽  
Standard Methods

AbstractTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.

scholarly journals Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro

Mutagenesis ◽  
2018 ◽  
Vol 33 (4) ◽  
pp. 283-289 ◽  
Author(s):  
Jatin R Verma ◽  
Danielle S G Harte ◽  
Ume-Kulsoom Shah ◽  
Huw Summers ◽  
Catherine A Thornton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymphoblastoid Cells ◽  
Imaging Flow Cytometry ◽  
Chemically Induced

scholarly journals The Biological Function of Extracellular Vesicles during Fertilization, Early Embryo—Maternal Crosstalk and Their Involvement in Reproduction: Review and Overview

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1510
Author(s):  
Emanuele Capra ◽  
Anna Lange-Consiglio
Keyword(s):  
Extracellular Vesicles ◽  
Spatial Interaction ◽  
Cell Communication ◽  
Early Embryo ◽  
Molecular Profile ◽  
Cell Of Origin ◽  
Non Invasive ◽  
Temporal And Spatial ◽  
Mediate Cell

Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.

scholarly journals Effect of Lidocaine and Epinephrine on Human Erythrocyte Shape and Vesiculability of Blood Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Tanja Slokar ◽  
Carlos Lopez-Mariscal ◽  
Judita Lea Krek ◽  
Roman Štukelj ◽  
Oskar Zupanc ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Whole Blood ◽  
Blood Cells ◽  
Statistical Power ◽  
Erythrocyte Shape ◽  
Plasma Lidocaine ◽  
Average Factor ◽  
Shape Changes

The effect of local anesthetic composed of lidocaine and epinephrine on vesiculability of blood cells and erythrocyte shape was studied. Whole blood and plasma were incubated with lidocaine/epinephrine. Extracellular vesicles were isolated by centrifugation and washing and counted by flow cytometry. Lidocaine/epinephrine and each component alone were added to diluted blood. Shape changes were recorded by micrographs. An ensemble of captured frames was analyzed for populations of discocytes, echinocytes, and stomatocytes by using statistical methods. Incubation of whole blood and blood plasma with lidocaine/epinephrine considerably increased concentration of extracellular vesicles in isolates (for an average factor 3.4 in blood and 2.8 in plasma). Lidocaine/epinephrine caused change of erythrocyte shape from mainly discocytic to mainly stomatocytic (higher than 50%). Lidocaine alone had even stronger stomatocytic effect (the percent of stomatocytes was higher than 95%) while epinephrine had echinocytic effect (the percent of echinocytes was higher than 80%). The differences were highly statistically significantp<10-8with statistical powerP=1. Lidocaine/epinephrine induced regions of highly anisotropically curved regions indicating that lidocaine and epinephrine interact with erythrocyte membrane. It was concluded that lidocaine/epinephrine interacts with cell membranes and increases vesiculability of blood cellsin vitro.

scholarly journals β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

2021 ◽  
Author(s):  
Zachary T Hilt ◽  
Preeti Maurya ◽  
Laura Tesoro ◽  
Daphne N Pariser ◽  
Sara K Ture ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
Elevated Plasma ◽  
Imaging Flow Cytometry ◽  
Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

scholarly journals High-throughput multicolor 3D localization in live cells by depth-encoding imaging flow cytometry

2019 ◽  
Author(s):  
Lucien E. Weiss ◽  
Yael Shalev Ezra ◽  
Sarah E. Goldberg ◽  
Boris Ferdman ◽  
Yoav Shechtman
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Imaging System ◽  
Live Cells ◽  
Flow Profile ◽  
Imaging Flow Cytometry ◽  
3D Localization ◽  
Large Populations ◽  
Point Spread Function Engineering

ABSTRACTImaging flow cytometry replaces the canonical point-source detector of flow cytometry with a camera, unveiling subsample details in 2D images while maintaining high-throughput. Here we show that the technique is inherently compatible with 3D localization microscopy by point-spread-function engineering, namely the encoding of emitter depth in the emission pattern captured by a camera. By exploiting the laminar-flow profile in microfluidics, 3D positions can be extracted from cells or other objects of interest by calibrating the depth-dependent response of the imaging system using fluorescent microspheres mixed with the sample buffer. We demonstrate this approach for measuring fluorescently-labeled DNA in vitro and the chromosomal compaction state in large populations of live cells, collecting thousands of samples each minute. Furthermore, our approach is fully compatible with existing commercial apparatus, and can extend the imaging volume of the device, enabling faster flowrates thereby increasing throughput.

scholarly journals Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

2020 ◽  
Author(s):  
Brian Jurgielewicz ◽  
Yao Yao ◽  
Steven L. Stice
Keyword(s):  
Flow Cytometry ◽  
Genetic Material ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Human Neural Stem Cells ◽  
Imaging Flow Cytometry ◽  
Gene Therapy Vectors ◽  
Time Variables

Abstract Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

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