Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Summary

We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.

Abstract

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Introduction

Small extracellular vesicles (sEVs) are nanosized, membrane-derived vesicles with a size range of 50-150 nm. They originate from multi-vesicular bodies (MVBs) and are released from cells upon fusion of the MVBs with the plasma membrane. sEVs contain miRNAs, mRNAs, proteins, and bioactive lipids, and these molecules are transferred between cells in the form of cell-to-cell communication. sEVs can be internalized by recipient cells by a variety of endocytic pathways, and this capture of sEVs by recipient cells is mediated by the recognition of surface molecules on both EVs and the target cells¹.

sEVs have gained intere....

Protocol

NOTE: All procedures were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care & Use Committee of Drexel University College of Medicine. Timed-pregnant CD-1 mice were used for astrocytic culture, and all dams were received 15 days after impregnation. Ten-twelve weeks old C57BL/6 mice were used for in vivo uptake experiments.

1. Isolation of sEVs from RAW 264.7 macrophage cells

1. Culture RAW 264.7 cells in 75 cm² flasks in DMEM exosome-depleted medium containing 10% exosome-depleted fetal bovine serum (FBS) and 1% penicillin-strep....

Results

After the isolation of sEVs from RAW 264.7 conditioned media via centrifugation, NTA was used to determine the concentration and size distribution of the purified sEVs. The average mean size of RAW 264.7-derived sEVs was 140 nm, and the peak particle size was 121.8 nm, confirming that most detectable particles in the light scattering measurement fell within the size range of exosomes or sEVs at 50-150 nm (Figure 1A). As suggested in the minimal information for studies of extracellular vesicl.......

Discussion

In this protocol, we showed the labeling of sEVs with PKH dyes and the visualization of their uptake in the spinal cord. PKH lipophilic fluorescent dyes are widely used for labeling cells by flow cytometry and fluorescent microscopy^{3,5,6,12,24,25}. Due to their relatively long half-life and low cytotoxicity, PKH dyes can be us.......

Materials

Name	Company	Catalog Number	Comments
Amicon Ultra 0.5 mL centrifugal filters	MilliporeSigma	Z677094
Anti-Alix Antibody	Abcam	ab186429	1:1000
Anti-Calnexin Antibody	Abcam	Ab10286	1:1000
Anti-CD81 Antibody	Santa Cruz Biotechnology	sc-166029	1:1000
Anti-GAPDH Monoclonal Antibody (14C10)	Cell Signaling Technology	2118	1:1000
Anti-Glial Fibrillary Acidic Protein Antibody	Sigma-Aldrich	MAB360	1:500 for IF; 1:1000 for IHC
Anti-Iba1 Antibody	Wako	019-19741	1:2000
Anti-MAP2A Antibody	Sigma-Aldrich	MAB378	1:500
Bovine Serum Albumin (BSA)	VWR	0332
Cell Strainer, 40 μm	VWR	15-1040-1
Centrifuge Tubes	Thermo Scientific	3118-0050	12,000 x g
Coverslip, 12-mm, #1.5	Electron Microscopy Sciences	72230-01
Coverslip, 18-mm, #1.5	Electron Microscopy Sciences	72222-01
DAPI	Sigma-Aldrich	D9542-1MG	1 µg/mL
DC Protein Assay	Bio-Rad	500-0116
Deoxyribonuclease I (DNAse I)	MilliporeSigma	D4513-1VL
Donkey Anti-Rabbit IgG H&L (HRP)	Abcam	ab16284	1:10000
Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488	Invitrogen	A-21206	1:500
Double Frosted Microscope Slides, #1	Thermo Scientific	12-552-5
DPBS without Calcium and Magnesium	Corning	21-031-CV
Dulbecco's Modified Eagle Medium (DMEM)	Corning	10-013-CV
Exosome-Depleted Fetal Bovine Serum	Gibco	A27208-01
Fetal Bovine Serum (FBS)	Corning	35-011-CV
FluorChem M imaging system	ProteinSimple
FV3000 Confocal Microscope	Olympus
Goat Anti-Mouse IgG H&L (HRP)	Abcam	ab6789	1:10000
Goat Anti-Mouse IgG H&L, Alexa Fluor 488	Invitrogen	A-11001	1:500
Goat Anti-Mouse IgG1, Alexa Fluor 594	Invitrogen	A-21125
Hank's Balanced Salt Solution (HBSS)	VWR	02-0121
HEPES	Gibco	15630080
HRP Substrate	Thermo Scientific	34094
Intercept blocking buffer, TBS	LI-COR Biosciences	927-60001
Laemmli SDS Sample Buffer	Alfa Aesar	AAJ61337AC
Micro Cover Glass, #1	VWR	48404-454
Microm HM550	Thermo Scientific
NanoSight NS300 system	Malvern Panalytical
NanoSight NTA 3.2 software	Malvern Panalytical
Neuro-2a Cell Line	ATCC	CCL-131
Normal Goat Serum	Vector Laboratories	S-1000
O.C.T Compound	Sakura Finetek	4583
Papain	Worthington Biochemical Corporation	NC9597281
Paraformaldehyde	Electron Microscopy Sciences	19210
Penicillin-Streptomycin	Gibco	15140122
PKH26	Sigma-Aldrich	MINI26-1KT
PKH67	Sigma-Aldrich	MINI67-1KT
Protease Inhibitor Cocktail	Thermo Scientific	1862209
PVDF Transfer Membrane	MDI	SVFX8302XXXX101
RAW 267.4 Cell Line	ATCC	TIB-71
RIPA Buffer	Sigma-Aldrich	R0278
Sodium Chloride	AMRESCO	0241-2.5KG
Superfrost Plus Gold Slides	Thermo Scientific	15-188-48	adhesive slides
T-75 Flasks	Corning	431464U
Tecnai 12 Digital Transmission Electron Microscope	FEI Company
TEM Grids	Electron Microscopy Sciences	FSF300-cu
Tris-Glycine Protein Gel, 12%	Invitrogen	XP00120BOX
Tris-Glycine SDS Running Buffer	Invitrogen	LC26755
Tris-Glycine Transfer Buffer	Invitrogen	LC3675
TrypLE Express		cell dissociation enzyme
Triton X-100	Acros Organics	327371000
Trypsin, 0.25%	Corning	25-053-CL
Tween 20
Ultracentrifuge Tubes	Beckman	344058	110,000 x g