Dataset Information

Development of a quantitative method to measure EV uptake.

ABSTRACT: The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.

SUBMITTER: Toribio V

PROVIDER: S-EPMC6642168 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a quantitative method to measure EV uptake.

Toribio Víctor V Morales Sara S López-Martín Soraya S Cardeñes Beatriz B Cabañas Carlos C Yáñez-Mó María M

Scientific reports 20190719 1

The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular loc ...[more]

PMID: 31324885

Similar Datasets

Project description:PURPOSE:Metabolism, and especially glucose uptake, is a key quantitative cell trait that is closely linked to cancer initiation and progression. Therefore, developing high-throughput assays for measuring glucose uptake in cancer cells would be enviable for simultaneous comparisons of multiple cell lines and microenvironmental conditions. This study was designed with two specific aims in mind: the first was to develop and validate a high-throughput screening method for quantitative assessment of glucose uptake in "normal" and tumor cells using the fluorescent 2-deoxyglucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and the second was to develop an image-based, quantitative, single-cell assay for measuring glucose uptake using the same probe to dissect the full spectrum of metabolic variability within populations of tumor cells in vitro in higher resolution. PROCEDURE:The kinetics of population-based glucose uptake was evaluated for MCF10A mammary epithelial and CA1d breast cancer cell lines, using 2-NBDG and a fluorometric microplate reader. Glucose uptake for the same cell lines was also examined at the single-cell level using high-content automated microscopy coupled with semi-automated cell-cytometric image analysis approaches. Statistical treatments were also implemented to analyze intra-population variability. RESULTS:Our results demonstrate that the high-throughput fluorometric assay using 2-NBDG is a reliable method to assess population-level kinetics of glucose uptake in cell lines in vitro. Similarly, single-cell image-based assays and analyses of 2-NBDG fluorescence proved an effective and accurate means for assessing glucose uptake, which revealed that breast tumor cell lines display intra-population variability that is modulated by growth conditions. CONCLUSIONS:These studies indicate that 2-NBDG can be used to aid in the high-throughput analysis of the influence of chemotherapeutics on glucose uptake in cancer cells.

Project description:BackgroundStrokes are a leading cause of disability worldwide, with many survivors experiencing difficulty in recovering upper extremity movement, particularly hand function and grasping ability. There is currently no objective measure of movement quality, and without it, rehabilitative interventions remain at best informed estimations of the underlying neural structures' response to produce movement. In this article, we utilize a novel modification to Procrustean distance to quantify curve dissimilarity and propose the Reach Severity and Dissimilarity Index (RSDI) as an objective measure of motor deficits.MethodsAll experiments took place at the Medstar National Rehabilitation Hospital; persons with stroke were recruited from the hospital patient population. Using Fugl-Meyer (FM) scores and reach capacities, stroke survivors were placed in either mild or severe impairment groups. Individuals completed sets of reach-to-target tasks to extrapolate kinematic metrics describing motor performance. The Procrustes method of statistical shape analysis was modified to identify reaching sub-movements that were congruous to able-bodied sub-movements.FindingsMovement initiation proceeds comparably to the reference curve in both two- and three-dimensional representations of mild impairment movement. There were significant effects of the location of congruent segments between subject and reference curves, mean velocities, peak roll angle, and target error. These metrics were used to calculate a preliminary RSDI score with severity and dissimilarity sub-scores, and subjects were reclassified in terms of rehabilitation goals as Speed Emphasis, Strength Emphasis, and Combined Emphasis.InterpretationThe modified Procrustes method shows promise in identifying disruptions in movement and monitoring recovery without adding to patient or clinician burden. The proposed RSDI score can be adapted and expanded to other functional movements and used as an objective clinical tool. By reducing the impact of stroke on disability, there is a significant potential to improve quality of life through individualized rehabilitation.

Dataset Information

Development of a quantitative method to measure EV uptake.

Publications

Development of a quantitative method to measure EV uptake.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets