Name: uptake of fluorescent labeled small extracellular vesicles in vitro and in spinal cord
Uploaded: 2021-05-23T14:00:00
Description: Watch this Scientific Journal Video about Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord at JoVE.com

This study was supported by grants from NIH NINDS R01NS102836 and the Pennsylvania Department of Health Commonwealth Universal Research Enhancement (CURE) awarded to Seena K. Ajit. We thank Dr. Bradley Nash for critical reading of the manuscript.

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 &#38; 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
<ol>
	<li>Culture RAW 26...

<ol>
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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 microscopy3,5,6,12,24,25. Due to their relatively long half-life and low cytotoxicity, PKH dyes can be us...

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 interest due to their capacity to trigger molecular and phenotypic changes in acceptor cells, their utility as a therapeutic agent, and their potential as carriers for cargo molecules or pharmacological agents. Due to their small size, the imaging and tracking of sEVs can be challenging, especially for in vivo studies and clinical settings. Therefore, many methods have been developed to label and image sEVs to assist their biodistribution and tracking in vitro and in vivo2.
The most common technique to study sEV biodistribution and target cell interactions involves labeling them with fluorescent dye molecules3,4,5,6,7. EVs were initially labeled with cell membrane dyes that were commonly used to image cells. These fluorescent dyes generally stain the lipid bilayer or proteins of interest on sEVs. Several lipophilic dyes display a strong fluorescent signal when incorporated into the cytosol, including DiR (1,1&#8242;-dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindotricarbocyanine iodide), DiL (1, 1&#8242;-dioctadecyl-3, 3, 3&#8242;, 3&#8242;-tetramethyl indocarbocyanine perchlorate), and DiD (1, 1&#8242;-dioctadecyl-3, 3, 3&#8242;, 3&#8242;-tetramethyl indocarbocyanine 4-chlorobenzenesulfonate salt)8,9,10,11.
Other lipophilic dyes, such as PKH67 and PKH26, have a highly fluorescent polar head group and a long aliphatic hydrocarbon tail that readily intercalates into any lipid structure and leads to long-term dye retention and stable fluorescence12. PKH dyes can also label EVs, which allows the study of EV properties in vivo13. Many other dyes have been used to observe exosomes using fluorescence microscopy and flow cytometry, including lipid-labeling dyes14 and cell-permeable dyes such as carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)15,16 and calcein acetoxymethyl (AM) ester17.
Studies of sEV-mediated crosstalk between different cells in the CNS have provided important insights on the pathogenesis of neuroinflammatory and neurodegenerative diseases18. For example, sEVs from neurons can spread beta-amyloid peptides and phosphorylated tau proteins and aid in the pathogenesis of Alzheimer&#39;s disease19. Additionally, EVs derived from erythrocytes contain large amounts of alpha-synuclein and can cross the blood-brain barrier and contribute to Parkinson&#39;s pathology20. The ability of sEVs to cross physiological barriers21 and transfer their biomolecules to target cells makes them convenient tools to deliver therapeutic drugs to the CNS22.
Visualizing sEV uptake by myriad CNS cells in the spinal cord will enable both mechanistic studies and the evaluation of the therapeutic benefits of exogenously administered sEVs from various cellular sources. This paper describes the methodology to label sEVs derived from macrophages and image their uptake in vitro and in vivo in the lumbar spinal cord by neurons, microglia, and astrocytes to qualitatively confirm sEV delivery by visualization.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amicon Ultra 0.5 mL centrifugal filters</td>
			<td>MilliporeSigma</td>
			<td>Z677094</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Alix Antibody</td>
			<td>Abcam</td>
			<td>ab186429</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Anti-Calnexin Antibody</td>
			<td>Abcam</td>
			<td>Ab10286</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Anti-CD81 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-166029</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Anti-GAPDH Monoclonal Antibody (14C10)</td>
			<td>Cell Signaling Technology</td>
			<td>2118</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>MAB360</td>
			<td>1:500 for IF; 1:1000 for IHC</td>
		</tr>
		<tr>
			<td>Anti-Iba1 Antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Anti-MAP2A Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>MAB378</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>0332</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer, 40 &#956;m</td>
			<td>VWR</td>
			<td>15-1040-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Thermo Scientific</td>
			<td>3118-0050</td>
			<td>12,000 x g</td>
		</tr>
		<tr>
			<td>Coverslip, 12-mm, #1.5</td>
			<td>Electron Microscopy Sciences</td>
			<td>72230-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip, 18-mm, #1.5</td>
			<td>Electron Microscopy Sciences</td>
			<td>72222-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-1MG</td>
			<td>1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Bio-Rad</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNAse I)</td>
			<td>MilliporeSigma</td>
			<td>D4513-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (HRP)</td>
			<td>Abcam</td>
			<td>ab16284</td>
			<td>1:10000</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Double Frosted Microscope Slides, #1</td>
			<td>Thermo Scientific</td>
			<td>12-552-5</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Exosome-Depleted Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A27208-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorChem M imaging system</td>
			<td>ProteinSimple</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FV3000 Confocal Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L (HRP)</td>
			<td>Abcam</td>
			<td>ab6789</td>
			<td>1:10000</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG H&#38;L, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1, Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-21125</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>VWR</td>
			<td>02-0121</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Substrate</td>
			<td>Thermo Scientific</td>
			<td>34094</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept blocking buffer, TBS</td>
			<td>LI-COR Biosciences</td>
			<td>927-60001</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli SDS Sample Buffer</td>
			<td>Alfa Aesar</td>
			<td>AAJ61337AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glass, #1</td>
			<td>VWR</td>
			<td>48404-454</td>
			<td></td>
		</tr>
		<tr>
			<td>Microm HM550</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300 system</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NTA 3.2 software</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuro-2a Cell Line</td>
			<td>ATCC</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington Biochemical Corporation</td>
			<td>NC9597281</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH67</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Thermo Scientific</td>
			<td>1862209</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Transfer Membrane</td>
			<td>MDI</td>
			<td>SVFX8302XXXX101</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW 267.4 Cell Line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>AMRESCO</td>
			<td>0241-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Gold Slides</td>
			<td>Thermo Scientific</td>
			<td>15-188-48</td>
			<td>adhesive slides</td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>Corning</td>
			<td>431464U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecnai 12 Digital Transmission Electron Microscope</td>
			<td>FEI Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM Grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FSF300-cu</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Glycine Protein Gel, 12%</td>
			<td>Invitrogen</td>
			<td>XP00120BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Glycine SDS Running Buffer</td>
			<td>Invitrogen</td>
			<td>LC26755</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Glycine Transfer Buffer</td>
			<td>Invitrogen</td>
			<td>LC3675</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td></td>
			<td>cell dissociation enzyme</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros Organics</td>
			<td>327371000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, 0.25%</td>
			<td>Corning</td>
			<td>25-053-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge Tubes</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>110,000 x g</td>
		</tr>
	</tbody>
</table>

uptake of fluorescent labeled small extracellular vesicles in vitro and in spinal cord

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.

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...

Watch this Scientific Journal Video about Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord at JoVE.com

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

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.

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

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...