Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Summary

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Abstract

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Introduction

Extracellular vesicles (EVs) are a population of small membrane-bound vesicles encapsulated with various proteins, lipids, and nucleic acids. Recent studies demonstrated that EVs play a crucial role in cell-cell communication, and are involved in the regulation of numerous physiological processes, including cell development, immune regulation, angiogenesis, and cell migration^2,3,4,5. Accumulating evidence indicates that EVs, circulating exosomes, and their miRNA cargo represents potential biomarkers of certain diseases⁶

Protocol

All animal experiments were approved by the local ethics committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit Number: SHVRIAU-14-0101).

1. In Vitro Culture of Schistosomes

Note: Schistosomes represent a biohazard. Workers should wear latex gloves at all times when handling worms, schistosomal suspensions, or other related biological materials. New Zealand rabbits were infected with ~2,000 cercariae via abdominal exposure. Schistosomes at the liver stage were collected from rabbits infected with S. japonicu....

Results

To quantify the yield of isolated SjEVs using the described protocol, we used BCA protein assay to access the protein concentration of the isolated SjEVs from 28-day adult schistosomes. As shown in Table 1, the SjEV protein concentration ranged from 208 µg to 250 µg per 100 mL of medium (Table 1).The particle size, as determined by Malvern nanoparticle analysis, indicated that the isolated SjEVs ranged from 100 nm to 400 nm, with the highest population ar.......

Discussion

Recent studies on EVs have demonstrated that schistosome EVs play an important role in host-pathogen interactions^3,9,12,16. To further address their regulatory functions, it is essential to isolate EVs from schistosomes. Here, we describe an alternative method for SjEV isolation. This method yields a wide range size of SjEVs, from 100 nm to 400 nm, in adult S. japonicum. The following .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Total Exosome Isolation Reagent (from cell culture media)	ThermoFisher SCIENTIFIC	4478359	For isolating S. japonicum extracellular vesicles
PKH67	Sigma-aldrich	MINI67	For labeling Evs
RPMI 1640 Medium	ThermoFisher SCIENTIFIC	11875119	For parasite culture
Penicillin-Streptomycin	ThermoFisher SCIENTIFIC	15140122
0.22μm syringe filter	Merck	SLGP033RB
SnakeSkin Dialysis Tubing	Thermo SCIENTIFIC	68035
PEG8000	Sangon Biotech	A100159
RIPA	Beyotime	P0013B
EMD Millipore™ Immobilon™ Western Chemiluminescent HRP Substrate (ECL)	Fisher scientific	WBKLS0100
DAPI	Cell Signaling Technology	4083
BCA kit	Beyotime	P0010
CD63 antibodies	Sangon Biotech	D160973-0025
PVDF	Bio-Rad	1704273
Formvar/Carbon 400 mesh	Ted Pella	01754-F
Phosphotungstic Acid	Ted Pella	19402
anti-mouse IgG-HRP	CWBIO	CW0102
NCTC clone 1469 cells	ATCC	ATCC® CCL-9.1™
FBS	HyClone	SV30087.02
Equipments
GE chemoluminescance imaging system	GE	ImageQuant LAS4000mini
Transmission electron microscopy	Hitachi	H-7600
Milli-Q water	Milli-Q	Milli-Q Elix
Eppendor Centrifuge	Eppendorf	Centrifuge 5804R
Zetasizer Nano	Malvern	Zetasizer Nano ZS
Ultracentrifuge	Beckman	Optima L-100 XP Ultracentrifuge
Incubator	ESCO	CCL-107B
Microscope	Zeiss	Zeiss Axin Observer Z1