Name: Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro (Video) | JoVE Journal
Uploaded: 2025-12-05T13:20:00.000+00:00
Duration: 10 min 40 s
Description: Learn to characterize adipocyte-derived extracellular vesicle secretion using a CD63-GFP reporter mouse model in vivo and in vitro. This method enables visualization and quantification of EV-mediated communication in adipose tissue.

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5 minutes

We thank the Einstein Flow Cytometry Core for FACS, and the Analytical Imaging Facility for all image analysis. This work was supported in part by National Institutes of Health grants DK128839 and NCI cancer center support grant P30CA013330.

In accordance with the animal care and use guidelines of Albert Einstein College of Medicine, male mice were group-housed at 22 &#176;C with 30-70% relative humidity on a 12 h light/12 h dark cycle, provided ad libitum access to food and water, and maintained on a C57BL/6J background.
1. In vivo detection of secreted AdEVs through uptake by APCs
<ol>
	<li>Buffer preparation
	<ol>
		<li>Digestion buffer: Prepare HEPES-buffered DMEM by adding 0.5 mL of 1 M HEPES and 0.5 g fatty acid-poor bovine serum albumin (BSA) to 50 mL of DMEM. Mix until dissolved.</li>
		<li>Wash buffer: Prepare PBS with 2.5 mM ethylenediaminetetraacetic acid (EDTA) and 4% fetal bovine serum (FBS).</li>
		<li>Prepare Liberase and DNase 1 stock at 5 mg/mL and at 5 KU/mL. Store at -20 &#176;C. Thaw immediately before use.</li>
	</ol>
	</li>
	<li>Inguinal adipose tissue collection and digestion 
	NOTE: Four-month-old AdipCD63-GFP and CD63-GFP control mice were euthanized via carbon dioxide inhalation. Before isolating APCs, euthanized mice were placed at 4 &#176;C for 24 h.
	<ol>
		<li>Excise adipose tissues using sterile technique and put 1 g of tissue in 0.5 mL of digestion buffer, and mince tissue finely using sterile scissors into pieces no larger than 1 mm.</li>
		<li>Suspend the minced tissue in 10 mL of digested buffer containing 0.5 mg/mL Liberase and 50 U/mL DNase 1.</li>
		<li>Incubate at 37 &#176;C on an orbital shaker (100 rpm) for 30-60 min. Gently mix the tissue by inverting the tube a few times during incubation. When the solution appears cloudy and no visible tissue fragments remain, the digestion is considered complete.</li>
	</ol>
	</li>
	<li>Isolation of APCs
	<ol>
		<li>Dilute the suspension with two volumes of the digestion buffer and gently invert the tube 3-4 times.</li>
		<li>Pass through a sterile 100 &#181;m filter to remove any undigested tissue.</li>
		<li>Centrifuge the filtrate at 300 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Discard the supernatant adipocyte part and label the pellet as the stromal vascular fraction (SVF).</li>
		<li>Prepare fresh 1&#215; red blood cell (RBC) lysis buffer with sterile water. Lyse the red blood cells in the SVF by incubating on ice for 5 min, protected from light.</li>
		<li>Add two volumes of wash buffer, then centrifuge filtrate at 400 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Follow the adipose tissue progenitor isolation kit (mouse) protocol, resuspend the SVF in 80 &#181;Lof buffer (PBS, pH 7.2, 0.5 % BSA, 2 mM EDTA) per gram of tissue.</li>
		<li>Add 20 &#181;L of non-adipocyte progenitor depletion cocktail per gram of tissue.</li>
		<li>Mix and incubate for 15 min at 2-8 &#176;C in the dark.</li>
		<li>Pass the sample through the LS column and collect the flow-through.</li>
		<li>Centrifuge the flow-through (negative for CD31, CD45, Ter119) at 300 &#215; g for 5 min at 4 &#176;C to obtain the APCs for the study.</li>
	</ol>
	</li>
	<li>Detection of secreted AdEVs through APC uptake by confocal microscopy
	<ol>
		<li>Seed 250 &#181;L of isolated APCs (1 &#215; 104 cells/mL) from AdipCD63-GFP or CD63-GFP mice onto collagen-coated 8-well chamber slides and allow cells to attach for approximately 6 h.</li>
		<li>Fix the cells with 4% paraformaldehyde for 10 min to preserve cellular and EV structures for imaging. 
		NOTE: In these assays, GFP signals mark AdEVs, and their detection within APCs by confocal microscopy indicates uptake of AdEVs by these progenitor cells.</li>
		<li>Wash the slides three times with PBS, mount with antifade medium, and capture GFP signals using a confocal microscope equipped with a 63&#215; oil-immersion objective. Acquire images as z-series stacks with a 0.5 &#181;m step size, using a 488 nm laser for GFP excitation and detecting emission at 500-550 nm.</li>
	</ol>
	</li>
	<li>Detection of secreted AdEVs through APC uptake by flow cytometry
	<ol>
		<li>Count APCs and dilute viability dye (e.g., Zombie) 1:1000 in 1X&#160;PBS. Suspend cells at 1&#160;&#215; 106 per 100 &#181;L. Incubate for 20 min on ice, protected from light.</li>
		<li>Wash cells once with wash buffer. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Suspend cells in brilliant staining buffer at 1 &#215; 106 cells per 100 &#181;L.</li>
		<li>Block nonspecific binding with Fc receptor blocking reagent (e.g., anti-CD16/CD32) 1:100 on ice for 10 min.</li>
		<li>Pre-spin Sca-1 antibody at 6,000 &#215; g for 10 min at 4 &#176;C. Then add 1 &#956;L of the Sca-1 antibody to the APC tube and incubate for 20 minutes on ice, keeping the tube covered with foil.</li>
		<li>Wash twice with 1 mL of wash buffer, centrifuge at 300 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Suspend cells in wash buffer at around 1 &#215; 106 cells per 500 &#181;L for flow cytometry sorting. Acquire data for Sca1+ and GFP+ cells on a spectral flow cytometer equipped with a 405 nm violet laser (450/50 nm filter for Pacific Blue) and a 488 nm blue laser (530/30 nm filter for GFP). Analyze the samples using the instrument&#39;s running software, with Sca1+ cells gated, and the GFP+ subset within this population analyzed, as shown in Figure 2.</li>
	</ol>
	</li>
</ol>
2. Detection and analysis of the secreted AdEVs in vitro
<ol>
	<li>Differentiation of APCs into mature adipocytes 
	NOTE: All cultures are maintained at 37 &#176;C, 8% CO2 unless otherwise stated.
	<ol>
		<li>Day 0: Coat dishes with 0.2% gelatin for 30 min at room temperature (RT), aspirate, and seed freshly isolated APCs in DMEM/F12 + 15% FBS + 1% Penicillin/Streptomycin (P/S) + 0.1% Primocin to reach 100% confluency before the differentiation.</li>
		<li>Day 1-8: Replace with MesenCult adipogenic differentiation medium (50 mL total: 45 mL of base + 5 mL of supplement + 0.5 mL of GlutaMAX + 50 &#181;L of Primocin), change to fresh differentiation medium on day 4.</li>
		<li>Day 9: Wash adipocytes twice with PBS, then replace with mature adipocyte culture medium (DMEM +1% BSA + 0.2% Primocin) and maintain for 16 h.</li>
	</ol>
	</li>
	<li>AdEV isolation and purification
	<ol>
		<li>Pass 15 mL adipocyte culture generated above, first through a 40 &#181;m filter and then a 30 &#181;m filter.</li>
		<li>Centrifuge the filtrate at 500 &#215; g for 5 min, then 2,000 &#215; g for 10 min to remove residual cells and debris.</li>
		<li>Pass the filtrate through a 0.8 &#181;m filter, then concentrate the medium from 15 mL to 0.5 mL using a 100 kDa cutoff ultrafiltration unit (15 mL sample volume) by centrifuging at 4,000 &#215; g for 15 min.</li>
		<li>Load sample onto size exclusion chromatography (SEC) columns with an automatic fraction collector and collect fractions F1-8.</li>
		<li>Concentrate the collected EV fractions using a 100 kDa cutoff ultrafiltration unit (4 mL sample volume) and proceed to verification of EV content of each fraction by western blot, nanoparticle tracking analysis instrument, and transmission electron microscopy (TEM).</li>
	</ol>
	</li>
	<li>AdEV validation, quantification, and size/morphology assessment
	<ol>
		<li>Western blot analysis of EV markers
		<ol>
			<li>Concentrate each fraction from 400 &#181;L to 25 &#181;L, then add 50 &#181;L of radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Denature the samples by adding 2&#215; sodium dodecyl sulfate (SDS) loading buffer and resolve proteins on a 4-12% tris-glycine gel.</li>
		</ol>
		</li>
		<li>Analysis of EV size, concentration, and quality using a nanoparticle tracking analysis instrument.
		<ol>
			<li>Before analysis, dilute the EV samples with sterile, 0.1 &#181;m filtered PBS at a 1:1000 ratio, ensuring the particle concentration falls within the optimal detection range (approximately 107-109 particles/mL). Gently mix the diluted samples to avoid bubble formation.</li>
			<li>Load the sample into the instrument equipped with a high-sensitivity scientific complementary metal-oxide-semiconductor (sCMOS) camera, a 532 nm green laser module, and a detection threshold of 10 nm (up to 1,000 nm)/ 106 to 109 particles/mL.</li>
			<li>Perform imaging and quantification using the NS Xplorer software suite, which applies a dynamic observation volume-based concentration algorithm.</li>
		</ol>
		</li>
		<li>Examination of EV morphology by transmission electron microscopy (TEM)
		<ol>
			<li>Submit 50 &#181;L of EVs, collected before concentration, to the core facility for negative staining of EVs. Apply EVs to carbon-only, 400-mesh copper grids that had been plasma-cleaned with a plasma cleaner.</li>
			<li>After brief adsorption, rinse the grids with distilled water, negatively stain with 1% (w/v) uranyl acetate, and remove excess stain with filter paper before air-drying. Then, image the grids using a transmission electron microscope operated at 120 KV.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Safety and waste information
<ol>
	<li>Handle paraformaldehyde and uranyl acetate in a fume hood with personal protective equipment (PPE), as both are toxic. Collect uranyl acetate waste in labeled hazardous containers.</li>
	<li>Treat all biological samples as potentially infectious and dispose of materials according to institutional biosafety policies and protocols approved by the Institutional Animal Care and Use Committee (IACUC).</li>
</ol>

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<li>Thery, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+information+for+studies+of+extracellular+vesicles+2018+%28MISEV2018%29%3A+A+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> J Extracell Vesicles. 7 (1), 1535750(2018).</li>
<li>Olson, A. L., Knight, J. B., Pessin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Syntaxin+4%2C+VAMP2%2C+and%2For+VAMP3%2Fcellubrevin+are+functional+target+membrane+and+vesicle+SNAP+receptors+for+insulin-stimulated+GLUT4+translocation+in+adipocytes%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.</a> Mol Cell Biol. 17 (5), 2425-2435 (1997).</li>
<li>Oh, E., Miller, R. A., Thurmond, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Syntaxin+4+overexpression+ameliorates+effects+of+aging+and+high-fat+diet+on+glucose+control+and+extends+lifespan%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Syntaxin 4 overexpression ameliorates effects of aging and high-fat diet on glucose control and extends lifespan.</a> Cell Metab. 22 (3), 499-507 (2015).</li>
<li>Merz, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enrichment+of+the+exocytosis+protein+STX4+in+skeletal+muscle+remediates+peripheral+insulin+resistance+and+alters+mitochondrial+dynamics+via+Drp1%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Enrichment of the exocytosis protein STX4 in skeletal muscle remediates peripheral insulin resistance and alters mitochondrial dynamics via Drp1.</a> Nat Commun. 13, 424(2022).</li>
<li>Yu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involution+of+brown+adipose+tissue+through+a+syntaxin+4+dependent+pyroptosis+pathway%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Involution of brown adipose tissue through a syntaxin 4 dependent pyroptosis pathway.</a> Nat Commun. 15, 2856(2024).</li>
<li>Verweij, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+exosome+secretion+from+single+cells+reveals+a+modulatory+role+for+GPCR+signaling%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.</a> J Cell Biol. 217 (3), 1129-1142 (2018).</li>
<li>Arora, A., Sharma, V., Gupta, R., Aggarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+extracellular+vesicles+derived+from+ex+vivo+culture+of+visceral+adipose+tissue%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">Isolation and characterization of extracellular vesicles derived from ex vivo culture of visceral adipose tissue.</a> Bio Protoc. 14 (11), e5011(2024).</li>
<li>Benedikter, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafiltration+combined+with+size+exclusion+chromatography+efficiently+isolates+extracellular+vesicles+from+cell+culture+media+for+compositional+and+functional+studies%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.</a> Sci Rep. 7, 15297(2017).</li>
<li>Nordin, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafiltration+with+size-exclusion+liquid+chromatography+for+high+yield+isolation+of+extracellular+vesicles+preserving+intact+biophysical+and+functional+properties%5BTitle%5D)+AND+%22Nanomedicine%22%5BJournal%5D)">Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.</a> Nanomedicine. 11 (4), 879-883 (2015).</li>
</ol>

The authors have no financial interest to declare.

The described protocol fills an important methodological gap by providing a robust and reproducible system to isolate and characterize AdEVs and their uptake by APCs. Compared to EVs from other sources, AdEVs are more lipid-rich and buoyant, making the classical ultracentrifugation-based EV isolation techniques less efficient35. However, most published protocols for AdEV purification still depend on ultracentrifugation, which can affect vesicle morphology, lead to EV aggregates, and cause protein ...

Adipose tissue is increasingly recognized as a dynamic endocrine organ with key roles in whole-body energy homeostasis1,2,3,4,5. Beyond its classical function as a lipid storage depot, adipocytes regulate systemic insulin sensitivity, and disturbances in adipose mass-whether excess or deficiency-are linked to insulin resistance and metabolic disease6,7,8,9. One mechanism proposed to contribute to this regulatory capacity is intercellular communication through secreted factors. While adipokines are well established as key endocrine signals from adipose tissue10,11,12,13,14,15,16,17,18, adipocytes also release a substantial number of extracellular vesicles (EVs), membrane-bound particles containing lipids, proteins, nucleic acids, and metabolites. Whether adipocyte-secreted EVs (AdEVs) also carry glycans, like other types of EVs, is still unclear19,20,21,22,23,24. Adipocytes have been proven to be a major source of EVs, as approximately 80% circulating exosomal microRNAs originate from adipocytes in mouse models, as demonstrated using adipocyte-specific Dicer knockout mice under baseline conditons25. The roles of AdEVs in metabolism, inflammation, insulin sensitivity, and adipogenesis have been reported, but remain incompletely understood26,27,28,29. Other potential functions, particularly in mediating crosstalk with other cell types and organs, are still largely unknown. Abundance26 of EVs suggests they could play important roles in both local and systemic signaling, but the biological significance and mechanisms of action of AdEVs are largely unexplored. This gap in knowledge highlights the need for robust and specialized methods to study AdEV secretion and function.
To address this gap, we generated adipocyte-specific CD63-GFP mice (AdipCD63-GFP) by crossing Adiponectin-Cre mice with Stopfl/fl/CD63-GFP mice (strain#:036865, JAX).&#160;Stopfl/fl/CD63-GFP littermates, which did not express CD63-GFP in the absence of Cre, were used as controls. The CD63-GFP reporter strain has been successfully used to trace cell type-specific EVs in vivo, including endothelial- and neuron-derived EVs30,31. Since Adiponectin is expressed only in mature adipocytes and not in adipocyte progenitor cells (APCs)32,33, and CD63 is a marker of EVs34, the GFP+ EVs detected in circulation and tissues can be specifically identified as AdEVs. As such, in this study, GFP+ EVs detected within APCs are interpreted as AdEVs that have been taken up by these progenitor cells. This reporter system allows direct monitoring of vesicle release and potential target interactions in vivo and in vitro, enabling the study of AdEVs within other cell types of adipose tissue. Importantly, AdEVs are particularly lipid-rich compared with EVs from other cell types, making them more buoyant, fragile, and challenging to recover with traditional ultracentrifugation methods35,36,37. High centrifugal forces can damage these vesicles or lead to significant sample loss and contamination, complicating downstream analysis. To overcome these limitations, we optimized a workflow that combines multiple low-speed centrifugation steps with size-exclusion chromatography, which minimizes shear stress, preserves vesicle integrity, and improves purity and yield, enabling accurate quantification and functional studies. Together, the CD63-GFP system and this optimized isolation pipeline provide an accessible and reproducible approach for investigating AdEV secretion and function in adipose tissue crosstalk and systemic metabolic regulation.

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Fisher</td><td>AM9260G</td><td>500x dilution</td></tr><tr><td>10x PBS</td><td>Fisher Scientific</td><td>70-013-032</td><td>1000x dilution with water</td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td>1000x dilution</td></tr><tr><td>Adipose Tissue Progenitor Isolation Kit, mouse</td><td>Miltenyi biotec</td><td>30-106-639</td><td>Ready for use</td></tr><tr><td>Amicon Ultra Centrifugal Filter, 100 kDa MWCO</td><td>Sigma</td><td>UFC9100</td><td>15 mL sample volume</td></tr><tr><td>Amicon Ultra Centrifugal Filter, 100 kDa MWCO</td><td>Sigma</td><td>UFC9100</td><td>4 mL sample volume</td></tr><tr><td>BSA</td><td>Sigma</td><td>A6003</td><td>Ready for use</td></tr><tr><td>Calnexin antibody</td><td>calnexin</td><td>ab22595</td><td>1000x dilution</td></tr><tr><td>CD63 antibody</td><td>abcam</td><td>ab217345</td><td>500x dilution</td></tr><tr><td>CD81</td><td>cell signaling Technology</td><td>10037s</td><td>500x dilution</td></tr><tr><td>Corning&amp;nbsp;BioCoat&amp;nbsp;Collagen I Culture Slide</td><td>Sigma</td><td>CLS354630</td><td>Ready for use</td></tr><tr><td>Corning&amp;nbsp;cell strainer</td><td>Sigma</td><td>CLS431750</td><td>40 &amp;mu;m pore size</td></tr><tr><td>Corning&amp;nbsp;cell strainer</td><td>Sigma</td><td>CLS431752</td><td>100 &amp;mu;m pore size</td></tr><tr><td>COX IV antibody</td><td>Cell Signaling Technology</td><td>4844</td><td>1000x dilution</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td>Ready for use</td></tr><tr><td>DMEM/F-12, GlutaMAX supplement</td><td>Thermofisher</td><td>10565018</td><td>Ready for use</td></tr><tr><td>Dnase 1</td><td>Sigma</td><td>D4513</td><td>Stock 5 KU/mL at -20 &amp;deg;C</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>100x dilution</td></tr><tr><td>Halt&amp;trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermofisher</td><td>78444</td><td>Ready for use</td></tr><tr><td>HSP70 antibody</td><td>abcam</td><td>ab181606</td><td>1000x dilution</td></tr><tr><td>Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species)</td><td>Fisher Scientific</td><td>50-112-9743</td><td>10x dilution in water</td></tr><tr><td>Izon Science Usa Ltd QEVORIGINAL 70NMGEN2 5/PK SDP</td><td>Fisher Scientific</td><td>NC2042225</td><td>Ready for use</td></tr><tr><td>LEICA STELLARIS 8 confocal microscope</td><td>Leica Microscopes</td><td>N/A</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Sigma</td><td>LIBTM-RO</td><td>Stock 5 mg/mL at -20 &amp;deg;C</td></tr><tr><td>Millex MCE syringe filter</td><td>Sigma</td><td>SLAA025</td><td>0.8 &amp;mu;m pore size</td></tr><tr><td>NanoSight Pro</td><td>Malvern Panalytical</td><td>N/A</td><td></td></tr><tr><td>Novex Tris-Glycine Mini Protein Gels, 4&amp;ndash;12%,</td><td>Thermofisher</td><td>XP04120BOX</td><td>Ready for use</td></tr><tr><td>Novex Tris-Glycine SDS Sample Buffer (2x)</td><td>Thermofisher</td><td>LC2676</td><td>Ready for use</td></tr><tr><td>Pacific Blue anti-mouse Ly-6A/E (Sca-1) Antibody</td><td>BioLegend</td><td>108119</td><td>25x dilution</td></tr><tr><td>Paraformaldehyde solution (16%)</td><td>Fisher Scientific</td><td>50-980-487</td><td>dilute to 4% with PBS</td></tr><tr><td>pluriStrainer S / 30 &amp;micro;m (Cell Strainer)</td><td>Fisher Scientific</td><td>NC0922459</td><td>30 &amp;mu;m pore size</td></tr><tr><td>Ponceau S solution</td><td>Sigma</td><td>P7170-1L</td><td>Ready for use</td></tr><tr><td>Primocin</td><td>Invivogen</td><td>NC9141851</td><td>500x dilution</td></tr><tr><td>ProLong Gold Antifade Reagent with DAPI</td><td>Cell Signaling Technology</td><td>8961S</td><td>Ready for use</td></tr><tr><td>Purified anti-mouse CD16/32 Antibody</td><td>BioLegend</td><td>101301</td><td>100x dilution</td></tr><tr><td>RIPA buffer&amp;nbsp;</td><td>Fisher Scientific</td><td>PI89900</td><td>10x dilution in water</td></tr><tr><td>Spectral flow cytometer (Aurora 3)</td><td>Cytek Biosciences</td><td>N/A</td><td></td></tr><tr><td>Syntaxin 4 antibody</td><td>Synaptic Systems</td><td>110042</td><td>5000x dilution</td></tr><tr><td>Tecnai 20 (Transmission Electron Microscope)</td><td>FEI</td><td>N/A</td><td></td></tr><tr><td>Tergeo-EM</td><td>PIE Scientific LLC</td><td>N/A</td><td></td></tr><tr><td>Vinculin antibody</td><td>abcam</td><td>ab18508</td><td>5000x dilution</td></tr></tbody></table>

characterization of adipocyte-derived extracellular vesicle secretion using a cd63-gfp reporter mouse model in vivo and in vitro

Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro

Adipocytes are increasingly recognized as active endocrine cells that secrete substantial quantities of EVs enriched in proteins, lipids, and nucleic acids. However, the mechanisms governing EV secretion and the dynamics of EV release in different biological contexts are not fully understood. To address this gap, we generated a novel adipocyte-specific CD63-GFP reporter mouse line, in which expression of GFP-tagged CD63 is driven by the Adiponectin promoter, enabling selective labeling and tracking of AdEVs in vivo. Using this reporter model, we examined secretion of AdEVs and their delivery to APCs within adipose tissues. Confocal imaging and flow cytometry revealed that AdEVs are actively taken up by APCs, highlighting a previously underappreciated mechanism of paracrine communication within the adipose microenvironment.
To complement the in vivo studies, an in vitro system was established, allowing for the direct monitoring of EV secretion from differentiated adipocytes. APCs were isolated from subcutaneous white adipose tissues of the reporter mice and induced to differentiate into mature adipocytes. In these cells, CD63-GFP signals were observed as discrete puncta and became enriched at the plasma membrane when EV secretion was inhibited, consistent with vesicle accumulation upon blocked release. Under normal growth conditions, EVs secreted into the culture medium were concentrated and subsequently purified using size exclusion chromatography, enabling downstream functional and biochemical analyses.
This platform can be applied to APCs derived from other adipose depots, such as epididymal white and brown adipose tissue, or by differentiating them into depot-specific adipocytes, thereby enabling comparisons of EV secretion among different adipocyte types.

Together, these approaches introduce a robust genetic tool and a complementary in vitro system to dissect the dynamics of AdEV secretion and targeting. These advances shed light on intra-adipose cellular communication and lay the groundwork for understanding the broader metabolic influence of AdEVs in health and disease.

Using this protocol of AdEVs detection through APC uptake by confocal microscopy, we isolated APCs from the inguinal adipose tissues of AdipCD63-GFP and Stopfl/fl/CD63-GFP mice and stained them with DAPI for visualization of the nuclei. When examined by confocal microscopy, APCs from Stopfl/fl/CD63-GFP mice showed no detectable GFP puncta (Figure 1A). In contrast, APCs isolated from AdipCD63-GFP mice exhibited numerous distinct GFP puncta throughout the cytoplasm (<stro...

The protocol uses an adipocyte-specific CD63-GFP reporter mouse line to visualize and quantify the secretion of adipocyte-derived extracellular vesicles (EVs) and demonstrate their uptake by progenitor cells, revealing a paracrine pathway within adipose tissue.

Adipocytes are increasingly recognized as active endocrine cells that secrete substantial quantities of EVs enriched in proteins, lipids, and nucleic ...

We study adipocyte-derived extracellular vesicle, EVs, to define their roles in adipose tissue, particularly how they regulate the crosstalk between mature adipocytes and their progenitor cells. There are currently no in vivo systems to study adipocyte EV secretions, and the existing purification protocols are not yet fully optimized. To begin, use sterile technique to excise inguinal adipose tissue from euthanized mice.Place one gram of tissue into a two milliliter tube containing 0.5 milliliters of digestion buffer. Using sterile scissors, mince the tissue into pieces no larger than one millimeter. Suspend the minced tissue in 10 milliliters of digestion buffer containing 0.5 milligrams per milliliter Liberase and 50 units per milliliter DNase I.Incubate the sample at 37 degrees Celsius on an orbital shaker.During incubation, gently invert the tube several times. When the solution appears cloudy and no visible tissue fragments remain, the digestion is complete. To isolate stromal vascular fraction or SVF, dilute the digested suspension with two volumes of digestion buffer.Gently invert the tube three to four times to mix well. Pass the suspension through a sterile 100-micrometer filter to remove undigested tissue, then centrifuge the filtrate at 300g for five minutes at four degrees Celsius. Label the pellet as SVF.Incubate the stromal vascular fraction in freshly prepared red blood cell lysis buffer on ice for five minutes, protected from light. Add two volumes of wash buffer and centrifuge at 400g for five minutes at four degrees Celsius. Resuspend the stromal vascular fraction in 80 microliters of buffer per gram of tissue and transfer the contents to a new tube.Add 20 microliters of non-adipocyte progenitor depletion cocktail per gram of tissue. Incubate the sample for 15 minutes at two to eight degrees Celsius in the dark. Pass the sample through an LS column and collect the flow through containing the lineage negative cell fraction.Centrifuge the flow through at 300g for five minutes at four degrees Celsius to obtain the adipocyte progenitor cells. Incubate 250 microliters of isolated mouse adipocyte progenitor cells mice onto collagen-coated eight-well chamber slides for six hours. Fix the cells with 4%paraformaldehyde for 10 minutes to preserve cellular and extracellular vesicle structures for imaging.Next, wash the slides three times with PBS, then mount with anti-fade medium. Next, after counting the adipocyte progenitor cells, suspend them and dilute viability dye. Incubate on ice for 20 minutes, protected from light.Wash the cells once with wash buffer and centrifuge. Suspend the cells in brilliant staining buffer so that the final concentration is 1 million cells per 100 microliters. Block nonspecific binding by adding Fc receptor blocking reagent at a one to 100 ratio and incubate.Free-spin the SCA-1 antibody at 6000g for 10 minutes at four degrees Celsius. Add the SCA-1 antibody to the APC tube at a one to 100 dilution, then place the tube on ice, protected from light. Wash the cells twice with one milliliter of wash buffer, then centrifuge at 300g for five minutes at four degrees Celsius.Suspend the cells in wash buffer for flow cytometry sorting, then acquire the data for SCA-1 positive and GFP positive cells on a spectral flow cytometer equipped with a violet and blue laser. To begin differentiation, coat dishes with 0.2%gelatin, for 30 minutes at room temperature. Seed freshly isolated APCs in supplemented DMEM/F12 medium and incubate until confluency.Replace the medium from day one to day eight with MesenCult adipogenic differentiation medium. On day nine, wash the adipocytes twice with PBS, then replace the medium with mature adipocyte culture medium containing DMEM 1%BSA and 0.2%Primocin. To isolate adipocyte-secreted extracellular vesicles, first pass 15 milliliters of culture medium through a 40-micrometer filter, followed by a 30-micrometer filter.Centrifuge the filtrate at 500g for five minutes then centrifuge the supernatant at 2000g for 10 minutes to remove any residual cells and debris. After passing the resulting supernatant through a 0.8-micrometer filter and transferring the filtrate into a 15-milliliter ultrafiltration unit, centrifuge at 4000g for 15 minutes to concentrate the medium. Load the concentrated sample onto a size exclusion chromatography column connected to an automatic fraction collector and collect fractions F1 through F8.To perform Western blot analysis, concentrate each fraction from 400 microliters to 25 microliters.Pipette 50 microliters of radio immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors to the concentrate. Add 75 microliters of two 2X SDS loading buffer to denature the sample and resolve proteins on a 4%to 12%Tris-Glycine gel. For nanoparticle tracking analysis, dilute extracellular vesicle samples with 0.1 micrometer filtered sterile PBS and mix gently.Load the diluted sample into the NTA instrument equipped with a high sensitivity scientific complementary metal oxide semiconductor camera and a 532-nanometer green laser. Set detection for particles between 10 nanometers and 1, 000 nanometers, and within a concentration of 10 to the power of six to 10 to the power of nine particles per milliliter. Adipose progenitor cells from stop-flock-flock/CD63GFP mice showed no detectable green fluorescent protein puncta under confocal microscopy.While cells from AdipCD63 mice displayed numerous distinct green fluorescent protein puncta throughout the cytoplasm. Flow cytometry analysis showed that 1.2%of skull 1 positive cells in the reporter mice expressed green fluorescent protein compared to 12.6%in AdipCD63-GFP mice. Western blotting of size exclusion chromatography fractions showed that extracellular vesicle markers CD63 and HSP70 were enriched in fractions F1 and F2.While markers of endoplasmic reticulum and mitochondria were absent, confirming vesical purity.Nanoparticle tracking analysis revealed that adipocyte-derived extracellular vesicles ranged in size from 50 nanometers to 500 nanometers, and transmission electron microscopy confirmed the vesicles exhibited intact double membrane structures. Western blot confirmed knockout of STX4 in adipocytes. Confocal microscopy showed that CD63-GFP positive vesicles were dispersed throughout the cytosol in wild type adipocytes, but accumulated near the plasma membrane in STX4 knockout adipocytes.The concentration of secreted adipocyte-derived extracellular vesicles was significantly reduced in STX4 knockout adipocytes compared to wild type. Western blot of equal protein amounts of intracellular vesicles showed reduced expression of CD63 and CD81 in STX4 knockout adipocytes compared to wild type. The protocol provides a robust and reproducible system to study adipocyte EV secretion in vivo and in vitro, as well as an optimized adipocyte EV purification method.The protocol for the first time describes and validates the use of serial low speed centrifugations combined with size exclusion chromatography to purify EVs derived from mature adipocytes. Our findings allow us to ask new questions about how adipocytes use extracellular vesicles to communicate with other cell types under physiological and pathological conditions.

characterization-adipocyte-derived-extracellular-vesicle-secretion

Research

JoVE Journal

Biology

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy.  Intravital microscopy is technically challenging in a moving organ such as the lung.   We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Using an ex vivo explant model, small pieces of the rostral portion of visceral white adipose tissue from lean mice are cultured for 7–10 days, enabling the induction and study of adipose tissue inflammation as it occurs during high-fat diet feeding.

An ex vivo Mouse Model for High-Fat Diet-Like Adipose Tissue Inflammation

Learn to characterize adipocyte-derived extracellular vesicle secretion using a CD63-GFP reporter mouse model in vivo and in vitro. This method enables visualization and quantification of EV-mediated communication in adipose tissue.

Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro (Video) | JoVE Journal

Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro

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