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

Protocol

1. EV isolation and on-chip immuno-fluorescent EV labeling

1. Collection of cell culture media (CCM) and pre-processing of CCM for EV isolation
   1. 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.
   2. Harvest the CCM.
   3. Centrifuge the CCM at 1000 x g for 10 min at room temperature (RT) to pellet any unattached cells and large d....

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.

Materials

Name	Company	Catalog Number	Comments
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