肝星状细胞中外泌体的分离、BODIPY标记和摄取

Shubham Shrivastava

doi:10.21769/BioProtoc.2633

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Peer-reviewed

Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells

肝星状细胞中外泌体的分离、BODIPY标记和摄取

Shubham Shrivastava email

发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2633 浏览次数: 12466

评审: Anonymous reviewer(s)

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Cover of Journal of Virology, featuring study using the protocol.

Mar 2017

Special Issue on Protocols for Organoid Research

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Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes by hepatic stellate cells.

Keywords: Exosomes (外泌体)

Hepatic stellate cells (肝星状细胞)

BODIPY (BODIPY)

Dynamic light scattering (动态光散射)

Hepatocytes (肝细胞)

Hepatitis C virus (丙型肝炎病毒)

Background

Exosomes are small membrane-bound extracellular vesicles of 40-150 nm in diameter. They have been identified in all body fluids, including urine, amniotic fluid, serum, saliva, breast milk, cerebrospinal fluid, nasal secretions and in the supernatant of tissue cultured cell lines. Exosomes mediate cell to cell communication through transfer of microRNAs (miRs), mRNAs, and proteins, etc. which can be taken up by neighboring or distant cells and subsequently promote signaling in recipient cells (Schorey et al., 2015). Exosomes are enriched in proteins, including members of the tetraspanin family (CD9, CD81, CD63), heat shock proteins (Hsp60, Hsp70, Hsp90) and proteins of the multivesicular bodies (annexins, RabGTPases and endosomal sorting complexes required for transport [ESCRT] proteins). Recent studies suggest that exosomes can influence immune response (Li et al., 2013; Sun et al., 2016), facilitate productive infection in naïve hepatoma cells (Ramakrishnaiah et al., 2013; Shrivastava et al., 2015), disease progression (Devhare et al., 2017) as well as drug resistance to anti-cancer therapy (Qu et al., 2016). Most commonly used techniques for isolation of exosomes involves ultracentrifugation (UC) and ExoQuick (EQ) precipitation. In hepatitis C virus (HCV) infection, crosstalk between liver resident cells including hepatocytes, macrophages, endothelial cells, lymphocytes and stellate cells play an important role in disease progression. We have shown earlier that HCV infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in the cell culture supernatants (Shrivastava et al., 2015). These exosomes carried miR-19a that induces fibrogenesis in hepatic stellate cells that might lead to liver fibrosis during HCV infection (Devhare et al., 2017). Here, we provide a protocol for labeling of exosomes with green fluorescent lipophilic dye, BODIPY that enables real-time monitoring and localization of exosomes within the recipient hepatic stellate cells.

Materials and Reagents

Serological pipettes (5 ml, 10 ml, 25 ml) (Corning)
50 ml polypropylene centrifuge tubes (Corning, catalog number: 430829 )
T-25 cm² cell culture flask (Corning, catalog number: 430639 )
100 mm tissue culture treated culture dish (Corning, catalog number: 430167 )
Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm for SW41Ti rotor (Beckman Coulter, catalog number: 344059 )
15 ml polypropylene centrifuge tubes (Corning, catalog number: 430791 )
4-well chambered slides (Thermo Fisher Scientific, Nunc, catalog number: 177437 )
Aluminium foil (Fisher Scientific, Fisherbrand^TM, catalog number: 01-213-102 )
0.2 μm size filter (Acrodisc Syringe Filters) (Pall, catalog number: 4612 )
Huh7.5 cell line (Human hepatocellular carcinoma cell line)
Rep2a-Rluc cell line: Huh7.5 cells harboring genome-length replicon of hepatitis C virus
LX2 cells (immortalized human hepatic stellate cells)
DMEM medium (Sigma-Aldrich, catalog number: D5796 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 16000044 )
Penicillin/streptomycin (Sigma-Aldrich, catalog number: P0781 )
Phosphate buffered saline (DPBS) without CaCl₂ and MgCl₂ (Sigma-Aldrich, catalog number: D8537 )
0.25% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
ExoQuick-TC (System Biosciences, catalog number: EXOTC10A-1 )
Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
4,6-Diamidino-2-phenylindole dihydrochloride(DAPI) (Sigma-Aldrich, catalog number: D9542 )
Alexa Fluor-647 conjugated wheat germ agglutinin (WGA) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: W32466 )
4,4-Difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: D3922 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
Formaldehyde solution (37 wt. % in H₂O) (Sigma-Aldrich, catalog number: 252549 )
TritonX-100 (Sigma-Aldrich, catalog number: T8787 )
Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H6648 )
BODIPY stock solution (see Recipes)
Exosome depleted serum (see Recipes)
Fixation solution (see Recipes)
Permeabilization buffer (see Recipes)
WGA conjugate stock solution (see Recipes)

Equipment

Pipette-aid
Water bath
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Sorvall ST 8R )
37 °C with 5% CO₂ cell culture incubator
Inverted microscope (Nikon Instruments, model: Eclipse TS100 , Objective: 10x)
SW41Ti rotor (Beckman Coulter, model: SW41Ti )
Zetasizer Nano (Malvern Instruments, model: NanoSight LM10 )
Olympus FV1000 confocal microscope (Olympus, model: FV1000 , Objective:60x)
Biosafety cabinet
Ultracentrifuge (Beckman Coulter, model: L8-80M )

Procedure

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Shrivastava, S. (2017). Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells. Bio-protocol 7(23): e2633. DOI: 10.21769/BioProtoc.2633.