Incucyte® EV Uptake Assay for Live-Cell Analysis
Exosomes and extracellular vesicles (EVs) are tiny membrane-bound vesicles that transport lipids, nucleic acids, and proteins between cells, contributing significantly to intercellular communication. They have emerged as key players in cell signaling, disease advancement, and therapeutic delivery, and have been shown to play a role in molecular mechanisms underlying cancer, neurodegenerative diseases, and cardiovascular disorders. Studies have shown the potential of EVs as highly valuable diagnostic and therapeutic tools (e.g. biomarkers or engineered to deliver precision medicines) due to ability to carry cargo of proteins, nucleic acids, and other molecules from their parent cells.
Given the significance of exosomes and EVs in various research areas, there is a growing need for reliable analytical methods to assess their uptake into live cells. Traditional methods for labeling EVs to track intercellular uptake are challenging, due to:
The Incucyte® Exofluor Green EV Labeling Kit, optimized for use with the Incucyte® Live-Cell Analysis System, enables real-time measurements of intracellular EV uptake within the same population of cells to monitor their biological functionality. With this kit, researchers can gain valuable insights into the mechanisms of EV uptake to help drive the development of future therapeutics.
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The Incucyte® Exofluor Green EV Labeling Kit enables non-perturbing, real-time, dynamic evaluation of cellular EV uptake and has been validated for use with the Incucyte® Live-Cell Analysis System configured with a Green | Red or Green | Orange | Near-IR optical module. Incucyte® Exofluor Green EV Labeling Dye uniformly labels the plasma membrane of small EVs (e.g., exosomes) by covalently binding to proteins and amino acids. The irreversible binding to the EVs - combined with the removal of free dye - mitigates the risk of non-specific dye attachment to other plasma membranes before or during uptake.
The Incucyte® Cell-by-Cell Analysis Software Module enables a range of applications for mixed cultures of adherent and non-adherent cells. Perform label-free cell counts on adherent and non-adherent cells and subsequent cell-by-cell classification based on shape, size or fluorescence intensity to quantify dynamic changes in subpopulations within a mixed culture, opening a new world of discovery.
Cat. No. 9600-0031
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The Incucyte® Live-Cell Analysis System is a powerful tool for researchers studying exosomes and EVs. Real-time image acquisition and analysis - coupled with an EV uptake assay - provide insight into dynamic cellular changes. Combining the EV uptake data with measurements of cell morphology, function and health yields valuable information about the effects of various treatments or culture conditions on EV uptake in a single well. Together, Incucyte® Live-Cell Analysis System, integrated software and Incucyte® Exofluor Green EV Labeling Kit provide a comprehensive approach that can greatly assist in the investigation of exosomes and EVs in live cells.
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Observe and evaluate changes in intracellular EV uptake or morphological changes in host cells over time on a population level
See example data below
Homogeneously labels EVs from various extraction methods and different source cells See example data below
Easy to follow assay set-up that preserves EVs and eliminates aggregation and free dyeSee example data below
Supports functional uptake of EVs and homogenous labeling for kinetic measurements or downstream analysisSee example data below
Figure 1. Uptake Cell Compatibility: A549 Exosomes (2 µg per well, HansaBioMed) were labeled using the Incucyte® Exofluor Dye. Uptake of labeled exosomes can be visualized across various cell types and media conditions (top row). Dye only control wells for each condition demonstrate the fluorescent signal is not due to residual free dye (bottom row). Images taken at 24-hours post-treatment with labeled A549-derived exosomes across various cell types show no change in cell morphology compared to controls.
Figure 2. EV Labeling Compatibility: Images demonstrate uptake of various EVs labeled with Incucyte® Exofluor Green by A549 recipient cells (24-hours post-treatment). A549 exosomes were extracted using ultrafiltration and SEC methods (HansaBioMed) and treated at 2 µg/well. Human plasma-derived exosomes (Abcam, ultrafiltration method of extraction) were used at a concentration of 4 µg/well. Both MSC and HEK293T EVs (Sartorius) were extracted using TFF/IEX chromatography and treated at 1.5 x 108 and 8.3 x 107 particles/well, respectively.
Figure 3. Quick guide of Incucyte® Exofluor Green EV Labeling Kit assay set up. Kit includes Vivaspin® columns for free dye removal and Minisart® filters to for sterilization and aggregate removal prior to imaging within the Incucyte® Live-Cell Analysis System.
Figure 4. EV Concentration Dependence: EVs were purified from HEK293T cells using ion exchange chromatography (Sartorius), labeled using the Incucyte® Exofluor Green EV Labeling Kit and added to A549 uptake cells. Phase and green fluorescent images were acquired every 2 hours over the course of 2 days and analyzed using Incucyte® Cell-by-Cell Software. (A) Labeled EVs were titrated ~40-fold (4.2 x 108 to 1 x 107 particles/well) and uptake was assessed using Average Green Mean Intensity. A corresponding concentration-dependent change in fluorescence was observed. (B) Phase Object Count (normalized to t=0) data illustrates no change in proliferation at all concentrations of EVs tested.
Figure 5. EV Uptake Depends on Endocytosis: A549 Cells were seeded overnight and then treated with the endocytosis inhibitor Dynasore (0.01 to 100 µM) in exosome-free culture medium. Immediately following Dynasore treatment, Hansa A549 exosomes labeled with Incucyte® Exofluor Green Dye (2 µg/well) were added. A concentration-dependent inhibition (IC50 = 2.72 µM) of exosome uptake was reflected in the intensity of green fluorescence (24-hour data shown).
Insights into Extracellular Vesicle Labeling and Cellular Uptake Using Live-Cell Analysis
The Incucyte® Exofluor Green EV Labeling Kit has been validated using EVs isolated from a variety of methods (including precipitation, ultrafiltration, TFF/ion exchange chromatography and ultracentrifugation).
Each column can process a maximum load of 40 µg protein or 100 µL labeled exosome sample. Higher protein loading or sample volumes may result in membrane clogging, and exceeding 100 µL labeled exosome sample will cause an increase in background fluorescence.
We recommend using exosomes within a range of 1-4 µg of protein/well or 5 x 107 - 1 x 109 particles/well.
The Incucyte® Exofluor Green EV Labeling Kit does not contain EVs/exosomes, only the labeling dye, Vivaspin® 2, and Minisart® filters. All EVs/Exosomes and other consumables must be supplied by the customer.
Our assay kit has been optimized and designed for use with the provided Vivaspin®2 columns and/or Minisart® filters, and differences in signal intensity and risk of perturbation has been observed with other consumables. If you require more columns or filters, please purchase from Sartorius (Catalog numbers provided in Product Guide, may differ based on pack size).
The Incucyte® EV Uptake Assay should be performed in Assay Media with exosome depleted serum (-eFBS) to minimize presence of endogenous exosomes that could impact uptake kinetics.
While there are three subclasses of EVs, this kit was validated using particles that were classified as exosomes. We have not tested labeling of microvesicles or apoptotic bodies.
We highly recommend using Cell-By-Cell Analysis. However, this assay can be performed using Basic Analyzer, but more optimization may be required. Please see Product Guide for more details.
The Incucyte® Exofluor Green EV Labeling Kit is intended for use in the Incucyte® SX5, S3, or SX1 Live-Cell Analysis Systems due to the green fluorescence signal.
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