Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

Published: April 30, 2023

Abstract

Source: Kim, C. J., et al., Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis. J. Vis. Exp. (2022).

In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell surface.

Protocol

1. EV isolation and on-chip immuno-fluorescent EV labeling Collection of cell culture media (CCM) and pre-processing of CCM for EV isolation Seed PC3 cells at 30% confluency in a 75 cm2 cell culture flask. Allow control cells to grow to 90% confluency (~48 h) in standard media and cell-line-specific supplements. NOTE: To prevent EV-containing components from affecting cellular uptake (i.e., fetal bovine serum), use exosome-depleted media and supplements. …

Representative Results

Figure 1: Schematic illustration of the EV isolation and on-chip labeling using a nano-filtration-based microfluidic device. (A) EVs isolation from CCM. (B) On-chip Immunofluorescent labeling of EVs. (C) Removal of unbound antibodies. <img alt="Figure 2" src="/file…

開示

The authors have nothing to disclose.

Materials

Alexa Fluor 488 anti-human CD63 Antibody Biolegend 353038 Fluorescent dye conjugated EV-specific antibody
CellTracker Orange CMTMR Dye Thermo Fisher Scientific C2927 Live cell (cytoplasm) fluoresent labeling reagent
CFI Apo Lambda S 40XC WI Nikon MRD77400 Objective for confocal imaging, NA=1.25
CFI Plan Apo VC 20X Nikon MRD70200 Objective for confocal imaging, NA=0.75
Exodisc Labspinner Inc. EX-D1001 A nano-filtration based microfluidic device for EV isolation
ExoDiscovery Labspinner Inc. EX-R1001 Operation device for Exodisc
Exosome-depleted FBS Thermo Fisher Scientific A2720801 Nutrient of cell culture media for PC3 cell line derived EV collection
Fetal bovine serum (FBS) VWR 1500-500 Nutrient for cell cultivation
Goat Anti-Mouse IgG H&L preadsorbed abcam ab7063 Mouse IgG antibody for negatvie control of EV labeling
Ibidi USA U DISH μ-Dish 35 mm Ibidi 81156 Culture dish for confocal imaging
Imaris 9.7.1 Oxford Instruments 9.7.1 Post-image processing software
Incubator System+ CO2/O2/N2 gas mixer Live Cell Instrument TU-O-20 Incubator system for live cell imaging
Nikon A1 HD25 / A1R HD25 camera Nikon NA Camera for confocal imaging
Nikon Eclipse Ti microscope Nikon NA Inverted microscope for confocal imaging
NIS-Elements AR 4.50.00 Nikon 4.50.00 Image processing software for Nikon microscope
NTA, NanoSight NS500 Malvern Panalytical NS500 Measurement device for EV concentration
OriginPro 2020 OriginLab 9.7.0.185 Graphing software
Penicillin-Streptomycin Thermo Fisher Scientific 15140122 Antibiotics for cell cultivation
RPMI 1640 Thermo Fisher Scientific 21875034 Cell culture media for PC3 cell line cultivation
SYTO RNASelect Green Fluorescent cell Stain – 5 mM Solution in DMSO Thermo Fisher Scientific S32703 RNA staining fluorescent dye for the EV labeling

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記事を引用
Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique. J. Vis. Exp. (Pending Publication), e21154, doi: (2023).

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