Journal: bioRxiv

Article Title: Immune cells regulate circulating adipocyte extracellular vesicle levels in response to metabolic shifts

doi: 10.1101/2025.07.11.664236

Figure Lengend Snippet: Validation of nano particle flow cytometry methodology. A. polystyrene microspheres of the indicated size. B . EVs were enriched from lean (normal chow) or obese (12 weeks high-fat diet) mouse plasma (2µl), with size exclusion chromatography (SEC) and analyzed by flow cytometry. The SSC detector was calibrated to display EV diameter C. CFSE stained EVs from lean and obese mice processed as in B. D. quantification of C E. A representative experiment where plasma is labeled with a cocktail of antibodies for EVs (CD63, CD81, ITGB1), or lipoproteins (APOE, APOB). EVs were enriched with SEC before flow analysis. F. Mouse plasma stained with an adipocyte marker mix (perilipin 1 and adiponectin). Plasma was harvested from either wild type mice or adipocyte-specific PPARγ knockout mice (Adipo-PPARγKO). G . Plasma stained from lean or obese (12 weeks high-fat diet). Total EVs that stain with the adipocyte marker mix (AdipoEVs) and EVs that co-stain with the both the adipocyte and EV marker mixes (Adipo/EV maker). H. Mitochondria isolated from mouse subcutaneous adipose tissue and stained with mitochondrial proteins: VDAC and COXIV or VDAC and TOM20. I. Plasma from mice fed a chow or high-fat diet (6 weeks) stained with markers for the indicated cell types. J. The percentage of EVs from each cell type in mouse plasma. Data are presented as mean ± s.e.m. * P < 0.05, *** P < 0.001

Article Snippet: Parameters were optimized using Flow Cytometry Sub-micron Particle Size Reference Kit (ThermoFIsher).

Techniques: Biomarker Discovery, Flow Cytometry, Clinical Proteomics, Size-exclusion Chromatography, Staining, Labeling, Marker, Knock-Out, Isolation

Journal: bioRxiv

Article Title: Immune cells regulate circulating adipocyte extracellular vesicle levels in response to metabolic shifts

doi: 10.1101/2025.07.11.664236

Figure Lengend Snippet: Circulating adipocyte EVs and EV-mitos are increased with high-fat feeding early and persistently. A. Body weights for a cohort of WT male and female (N=6 for each sex and group). B. Nano particle flow cytometry for lipoprotein markers in mice plasma (APOB/E antibodies). Mice were on diets for 6 weeks. C-D Total EVs in plasma (CFSE labeled) were quantified at day 0 and day 1 of the feeding experiment ( C ) or at baseline, and 30 or 60 minutes after bleeding ( D ). Plasma as collected from male and female cohorts (N=6) via tail vein bleed over the indicated time course after diet initiation (chow or high-fat diet; HFD). E-L EVs were analyzed for total EVs (CFSE-stained; E-F ), adipocyte EVs (adipoEVs; APN/PLN1 + ; G-H ), adipocyte EV-mitos (adipoEV-mitos; APN/PLN1 + , VDAC + ; I-J ), and total mitochondria (VDAC + , K-L ). E, G, I and K are acute timepoints. F, H, J, L are chronic timepoints. Data are presented as mean ± s.e.m. * P < 0.05, *** P < 0.001.

Article Snippet: Parameters were optimized using Flow Cytometry Sub-micron Particle Size Reference Kit (ThermoFIsher).

Techniques: Flow Cytometry, Clinical Proteomics, Labeling, Staining

Journal: bioRxiv

Article Title: Immune cells regulate circulating adipocyte extracellular vesicle levels in response to metabolic shifts

doi: 10.1101/2025.07.11.664236

Figure Lengend Snippet: AdipoEVs correlate with insulin resistance in people with obesity. Plasma samples from metabolically healthy lean (MHL), metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO) patients were analyzed by nano flow cytometry for A. Lipoproteins (APOE/B + ), B. total EVs (CFSE + ), C. adipoEVs (APN/PLN1 + ), D. adipoEV-mitos (APN/PLN1 + , VDAC + ), E. total mitochondria (VDAC + ), F. Adipose tissue (AT) immune cell EVs (APN/PLN + , CD45 + ), G. total immune cell EVs (CD45 + ), or H. Endothelial cell (EC) EVs (CD31 + /CD45 - ). AdipoEVs correlations with I. % fat mass J. insulin sensitivity, K. hepatic insulin sensitivity index (HISI), L. homeostatic model assessment of insulin resistance (HOMA-IR), M. Intrahepatic triglyceride (IHTC) content. Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Parameters were optimized using Flow Cytometry Sub-micron Particle Size Reference Kit (ThermoFIsher).

Techniques: Clinical Proteomics, Metabolic Labelling, Flow Cytometry

Journal: bioRxiv

Article Title: Immune cells regulate circulating adipocyte extracellular vesicle levels in response to metabolic shifts

doi: 10.1101/2025.07.11.664236

Figure Lengend Snippet: Macrophage-mediated clearance of EVs is reduced with high-fat feeding. A. AdipoEVs (APN/PLN1 + ) correlate with total EVs (CFSE + ). B. Timeline of clodronate experiment. Mice were on a chow of high fat diet for 6 weeks at which point 5µl/gram mouse body weight of either control liposomes or clodronate liposomes were injected into mice at day 0 and day 3. Blood was sampled at the indicated timepoints. At day 4 of macrophage depletions and fed/fasted study was conducted. At day 6, mice were injected i.v. with purified plasma or adipocyte EVs labeled with CFSE for determination of EV clearance rates. Total EVs (CFSE + ; C ) and adipoEVs ( D ) were quantified at the indicated timepoints. At day 2 post clodronate injection plasma samples were analyzed for E. hepatocyte EVs (ASGR1 + /CD45 - ), F. endothelial cell (EC) EVs (CD31 + /CD45 - ), G. myocyte EVs (MHC + , CD45 + ). H. platelet EVs (CD41 + ), I. red blood cell (RBC) EVs (TER119 + ), or J . lipoproteins (APOE/B + ). K-M Mouse plasma EVs were harvested, labeled with CFSE, purified by SEC and injected retro-orbitally into mice on a chow or high fat diet (HFD) either treated with empty liposomes (Control) or clodronate liposomes (Clod). Tail blood was sampled at the indicated timepoints following CFSE + EV injection and the CFSE + EV remaining in the blood was quantified. N-O Mice treated with empty liposomes (Control) or clodronate liposomes were either ad libitum fed or fasted for 16 hours and refed for 3 hours. Plasma was sampled under each metabolic state and total EVs (CFSE; N ) and adipoEVs ( O ) were quantified. At day 7 of the experiments (4 days after the last clodronate injection) mice were euthanized. P. Macrophages in the eWAT and Q. monocytes in the blood were quantified by flow cytometry in mice treated with control liposomes (Cntrl) or clodronate liposomes (Clod). R. EV clearance assay for adipocyte-derived CFSE + EVs, as described in K-M. S. Blood monocyte uptake of adipocyte-derived CFSE + EVs at the indicated timepoints after EV injection by flow cytometry. Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Parameters were optimized using Flow Cytometry Sub-micron Particle Size Reference Kit (ThermoFIsher).

Techniques: Control, Liposomes, Injection, Purification, Clinical Proteomics, Labeling, Flow Cytometry, Derivative Assay