Extracellular Vesicle Uptake Assay <em>via</em> Confocal Microscope Imaging Analysis

Chi-Ju Kim; Morgan D. Kuczler; Liang Dong; Junyoung Kim; Sarah R. Amend; Yoon-Kyoung Cho; Kenneth J. Pienta

doi:10.3791/62836

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

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Biology

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Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

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08:32 min

February 14, 2022

DOI: 10.3791/62836-v

Chi-Ju Kim*^1,2, Morgan D. Kuczler*¹, Liang Dong^1,3, Junyoung Kim^2,4, Sarah R. Amend¹, Yoon-Kyoung Cho^2,4, Kenneth J. Pienta¹

¹The Brady Urological Institute,Johns Hopkins University School of Medicine, ²Department of Biomedical Engineering, School of Life Sciences,Ulsan National Institute of Science and Technology (UNIST), ³Department of Urology, Renji Hospital,Shanghai Jiao Tong University School of Medicine, ⁴Center for Soft and Living Matter,Institute for Basic Science (IBS)

Overview

This study addresses the need for effective assays to visualize and quantify the uptake of extracellular vesicles (EVs) by cells. Utilizing a nano-filtration-based microfluidic device and 3D fluorescence imaging via confocal microscopy, the protocol enables the analysis of EV uptake, which can provide insights into intercellular communication.

Key Study Components

Research Area

Extracellular vesicles (EVs)
Intercellular communication
Cancer biology and drug delivery

Background

EVs play a crucial role in cellular biology and communication.
There is a need for practical methodologies to measure EV uptake.
Understanding EV dynamics can aid in therapeutic applications.

Methods Used

3D fluorescence imaging via confocal microscopy
PC3 cells as a model system
Nano-filtration-based microfluidic device for EV isolation

Main Results

Quantification of internalized EVs using volumetric analysis.
Demonstrated that the uptake level depends on incubation time.
Systematic exclusion of non-internalized EVs enhances accuracy.

Conclusions

The study provides a reliable protocol for analyzing EV uptake.
This method has implications for studies in cancer therapy and drug delivery.

Frequently Asked Questions

What are extracellular vesicles?

Extracellular vesicles (EVs) are membrane-bound particles released by cells that facilitate intercellular communication by transferring biomolecules.

Why is EV uptake important?

EV uptake is critical for understanding how cells communicate and can influence therapeutic strategies, especially in cancer research.

What technologies are used in this protocol?

The protocol employs confocal microscopy for imaging and a microfluidic device for EV isolation.

How do you visualize internalized EVs?

Internalized EVs are labeled with fluorescent antibodies, allowing for visualization under a confocal microscope.

What cell line is used in this research?

PC3 cells, a prostate cancer cell line, are utilized for the EV uptake assays.

Is this method applicable to other types of cells?

Yes, the protocol can potentially be adapted to analyze EV uptake in various cell types based on research needs.

What biological implications does EV uptake have?

Understanding EV uptake can provide insights into disease mechanisms, therapeutic delivery, and cellular interactions in various biological contexts.

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

This protocol provides a robust methodology for analyzing EVs at a cellular level, and a practical approach for efficient EV uptake and EV tracking analysis. This method of EV labeling eliminates co-precipitation of EVs and dye, thus enhancing the EV signals. EV uptake is measured by volumetric analysis to distinguish the internalized EVs from the superficial EVs.

EVs are active cargo, thus, their uptake allows the transfer of biochemical molecules. For research on EV-based drug delivery and cancer therapy, this technique can be an assessment tool. EV uptake plays a role in intercellular communication and is investigated in various research fields, such as cancer biology, neuroscience, and drug delivery.

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