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Method Article

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

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DOI:

10.3791/69670

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December 5th, 2025

 ,  ,  ,  ,  ,  ,  , 
1Department of Medicine, Albert Einstein College of Medicine, 2Fleischer Institute for Diabetes and Metabolism, Albert Einstein College of Medicine, 3Department of Molecular Pharmacology, Albert Einstein College of Medicine, 4Department of Microbiology and Immunology, Albert Einstein College of Medicine, 5Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, 6Department of Developmental and Molecular Biology, Albert Einstein College of Medicine

In This Article

Summary

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.

Abstract

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.

Introduction

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). 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.

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Protocol

In accordance with the animal care and use guidelines of Albert Einstein College of Medicine, male mice were group-housed at 22 °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

  1. Buffer preparation
    1. 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.
    2. Wash buffer: Prepare PBS with 2.5 mM ethylenediaminetetraacetic acid (EDTA) and 4% fetal bovine serum (FBS).
    3. Prepare Liberase and DNase 1 stock at 5 mg/mL and at 5 KU/mL. Store at -20 °C. Thaw immediately before use.
  2. 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 °C for 24 h.
    1. 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.
    2. Suspend the minced tissue in 10 mL of digested buffer containing 0.5 mg/mL Liberase and 50 U/mL DNase 1.
    3. Incubate at 37 °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.
  3. Isolation of APCs
    1. Dilute the suspension with two volumes of the digestion buffer and gently invert the tube 3-4 times.
    2. Pass through a sterile 100 µm filter to remove any undigested tissue.
    3. Centrifuge the filtrate at 300 × g for 5 min at 4 °C.
    4. Discard the supernatant adipocyte part and label the pellet as the stromal vascular fraction (SVF).
    5. Prepare fresh 1× 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.
    6. Add two volumes of wash buffer, then centrifuge filtrate at 400 × g for 5 min at 4 °C.
    7. Follow the adipose tissue progenitor isolation kit (mouse) protocol, resuspend the SVF in 80 µLof buffer (PBS, pH 7.2, 0.5 % BSA, 2 mM EDTA) per gram of tissue.
    8. Add 20 µL of non-adipocyte progenitor depletion cocktail per gram of tissue.
    9. Mix and incubate for 15 min at 2-8 °C in the dark.
    10. Pass the sample through the LS column and collect the flow-through.
    11. Centrifuge the flow-through (negative for CD31, CD45, Ter119) at 300 × g for 5 min at 4 °C to obtain the APCs for the study.
  4. Detection of secreted AdEVs through APC uptake by confocal microscopy
    1. Seed 250 µL of isolated APCs (1 × 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.
    2. 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.
    3. Wash the slides three times with PBS, mount with antifade medium, and capture GFP signals using a confocal microscope equipped with a 63× oil-immersion objective. Acquire images as z-series stacks with a 0.5 µm step size, using a 488 nm laser for GFP excitation and detecting emission at 500-550 nm.
  5. Detection of secreted AdEVs through APC uptake by flow cytometry
    1. Count APCs and dilute viability dye (e.g., Zombie) 1:1000 in 1X PBS. Suspend cells at 1 × 106 per 100 µL. Incubate for 20 min on ice, protected from light.
    2. Wash cells once with wash buffer. Centrifuge at 300 × g for 5 min at 4 °C.
    3. Suspend cells in brilliant staining buffer at 1 × 106 cells per 100 µL.
    4. Block nonspecific binding with Fc receptor blocking reagent (e.g., anti-CD16/CD32) 1:100 on ice for 10 min.
    5. Pre-spin Sca-1 antibody at 6,000 × g for 10 min at 4 °C. Then add 1 μL of the Sca-1 antibody to the APC tube and incubate for 20 minutes on ice, keeping the tube covered with foil.
    6. Wash twice with 1 mL of wash buffer, centrifuge at 300 × g for 5 min at 4 °C.
    7. Suspend cells in wash buffer at around 1 × 106 cells per 500 µL 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's running software, with Sca1+ cells gated, and the GFP+ subset within this population analyzed, as shown in Figure 2.

2. Detection and analysis of the secreted AdEVs in vitro

  1. Differentiation of APCs into mature adipocytes
    NOTE: All cultures are maintained at 37 °C, 8% CO2 unless otherwise stated.
    1. 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.
    2. 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 µL of Primocin), change to fresh differentiation medium on day 4.
    3. 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.
  2. AdEV isolation and purification
    1. Pass 15 mL adipocyte culture generated above, first through a 40 µm filter and then a 30 µm filter.
    2. Centrifuge the filtrate at 500 × g for 5 min, then 2,000 × g for 10 min to remove residual cells and debris.
    3. Pass the filtrate through a 0.8 µm 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 × g for 15 min.
    4. Load sample onto size exclusion chromatography (SEC) columns with an automatic fraction collector and collect fractions F1-8.
    5. 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).
  3. AdEV validation, quantification, and size/morphology assessment
    1. Western blot analysis of EV markers
      1. Concentrate each fraction from 400 µL to 25 µL, then add 50 µL of radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Denature the samples by adding 2× sodium dodecyl sulfate (SDS) loading buffer and resolve proteins on a 4-12% tris-glycine gel.
    2. Analysis of EV size, concentration, and quality using a nanoparticle tracking analysis instrument.
      1. Before analysis, dilute the EV samples with sterile, 0.1 µm 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.
      2. 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.
      3. Perform imaging and quantification using the NS Xplorer software suite, which applies a dynamic observation volume-based concentration algorithm.
    3. Examination of EV morphology by transmission electron microscopy (TEM)
      1. Submit 50 µL 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.
      2. 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.

3. Safety and waste information

  1. 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.
  2. 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).

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Results

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 (

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Discussion

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 ...

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Disclosures

The authors have no financial interest to declare.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.5 M EDTA, pH 8.0FisherAM9260G500x dilution
10x PBSFisher Scientific70-013-0321000x dilution with water
1 M HEPESGibco156300801000x dilution
Adipose Tissue Progenitor Isolation Kit, mouseMiltenyi biotec30-106-639Ready for use
Amicon Ultra Centrifugal Filter, 100 kDa MWCOSigmaUFC910015 mL sample volume
Amicon Ultra Centrifugal Filter, 100 kDa MWCOSigmaUFC91004 mL sample volume
BSASigmaA6003Ready for use
Calnexin antibodycalnexinab225951000x dilution
CD63 antibodyabcamab217345500x dilution
CD81cell signaling Technology10037s500x dilution
Corning BioCoat Collagen I Culture SlideSigmaCLS354630Ready for use
Corning cell strainerSigmaCLS43175040 μm pore size
Corning cell strainerSigmaCLS431752100 μm pore size
COX IV antibodyCell Signaling Technology48441000x dilution
DMEMGibco11965092Ready for use
DMEM/F-12, GlutaMAX supplementThermofisher10565018Ready for use
Dnase 1SigmaD4513Stock 5 KU/mL at -20 °C
GlutaMAX SupplementGibco35050061100x dilution
Halt™ Protease and Phosphatase Inhibitor Cocktail (100X)Thermofisher78444Ready for use
HSP70 antibodyabcamab1816061000x dilution
Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species)Fisher Scientific50-112-974310x dilution in water
Izon Science Usa Ltd QEVORIGINAL 70NMGEN2 5/PK SDPFisher ScientificNC2042225Ready for use
LEICA STELLARIS 8 confocal microscopeLeica MicroscopesN/A
Liberase TM Research GradeSigmaLIBTM-ROStock 5 mg/mL at -20 °C
Millex MCE syringe filterSigmaSLAA0250.8 μm pore size
NanoSight ProMalvern PanalyticalN/A
Novex Tris-Glycine Mini Protein Gels, 4–12%,ThermofisherXP04120BOXReady for use
Novex Tris-Glycine SDS Sample Buffer (2x)ThermofisherLC2676Ready for use
Pacific Blue anti-mouse Ly-6A/E (Sca-1) AntibodyBioLegend10811925x dilution
Paraformaldehyde solution (16%)Fisher Scientific50-980-487dilute to 4% with PBS
pluriStrainer S / 30 µm (Cell Strainer)Fisher ScientificNC092245930 μm pore size
Ponceau S solutionSigmaP7170-1LReady for use
PrimocinInvivogenNC9141851500x dilution
ProLong Gold Antifade Reagent with DAPICell Signaling Technology8961SReady for use
Purified anti-mouse CD16/32 AntibodyBioLegend101301100x dilution
RIPA buffer Fisher ScientificPI8990010x dilution in water
Spectral flow cytometer (Aurora 3)Cytek BiosciencesN/A
Syntaxin 4 antibodySynaptic Systems1100425000x dilution
Tecnai 20 (Transmission Electron Microscope)FEIN/A
Tergeo-EMPIE Scientific LLCN/A
Vinculin antibodyabcamab185085000x dilution

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Adipocyte EVsExtracellular VesiclesEV SecretionStromal Vascular FractionAdipocyte Progenitor CellsSize Exclusion ChromatographyFlow CytometryConfocal MicroscopyNanoparticle Tracking
Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro
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