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The authors thank Constance Hobusch and Florian Kirmse for excellent technical assistance. Flow cytometry analyses were performed at the Core Unit Fluorescence Technologies, Leipzig University, with technical support from Kathrin J&#228;ger and Pia Gl&#246;ckner. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Project Number 209933838 &#8211; SFB 1052 (Project B09).

Animal experiments were performed in accordance with institutional and national guidelines and were approved by the appropriate regulatory authorities. Csf1r-eGFP-Adipoq-ERT2-Cre-CAG-tdTomato mice ("MacFat", RRID: Csf1r-eGFP: RRID:IMSR_JAX:018549, Adipoq-iCreERT2: RRID:IMSR_JAX:025124, CAG-tdTomato: RRID:IMSR_JAX:007914)27,28 were used for representative microscopy experiments. This reporter line enables visualization of macrophages by GFP expression and adipocytes by tdTomato expression. Because tdTomato expression is lost following adipocyte death, adipocyte viability can be monitored during culture. Csf1r-eGFP reporter mice ("MacGreen", RRID:IMSR_JAX:018549)28 were used for macrophage visualization and flow cytometry analyses. C57BL/6J mice (RRID:IMSR_JAX:000664) were used for non-imaging experiments, including scRNA-seq and qPCR analyses. All transgenic lines were maintained as heterozygous mice on a C57BL/6J background.
Animal experiments followed the ‘Principles of laboratory animal care’ (NIH publication no. 85e23, revised 1985) and specific national laws and were approved by the local authorities of the state of saxony: Landesdirektion in Leipzig, Germany (animal experimental application: TV11-21-MEZ). According to local authorities, the experiments described do not constitute animal testing, as it states “if a vertebrate is killed solely for the purpose of using its organs or tissues for scientific purposes, this is not considered animal testing under § 7(2) of the Animal Welfare Act (TierSchG), but rather killing under § 4(3) of the Animal Welfare Act (TierSchG).” The killing of the animals must be reported in an ‘animal report.’ This report is included in the animal experimentation application. The ethics committee of the Faculty of Medicine at the University of Leipzig has also approved the research projects. In accordance with the guidelines of animal experimental application TV11-21-MEZ, the mice are euthanized by an overdose of the inhalation anesthetic isoflurane. An effective gas concentration (5% in O₂ carrier gas) is delivered via an isoflurane vaporizer. The euthanasia procedure is complete when definitive circulatory arrest (absence of breathing [feather test] and heartbeat [palpation]) is confirmed.
Mice were housed in a temperature-controlled (22 ± 2 °C), specific pathogen-free facility at Leipzig University in sibling groups of 3–5 animals under a 12 h light/dark cycle. Animals had ad libitum access to water and a standard chow diet (9% kcal fat; Sniff GmbH, Germany). Representative experiments were performed using male and female mice. The reagents, chemicals, and kits used in the protocol are listed in the Table of Materials.
1. Preparations
<ol>
	<li>Prepare reagents and equipments
	<ol>
		<li>Prepare the culture medium under sterile conditions by supplementing RPMI-1640 with 10% fetal bovine serum (FBS) and 1% antibiotic solution.</li>
		<li>Warm the culture medium and sterile PBS to 37 °C.</li>
		<li>Clean all equipment and the work area inside the tissue culture hood with 70% ethanol.</li>
	</ol>
	</li>
	<li>Prepare animals
	<ol>
		<li>Sacrifice mice according to institutional guidelines. Optionally weigh the animals.</li>
		<li>Place the animals in a glass beaker containing 70% ethanol.</li>
		<li>Wipe the exterior of the glass beaker with ethanol and transfer it into the tissue culture hood.</li>
	</ol>
	</li>
</ol>
2. Adipose tissue isolation (Figure 2)
<ol>
	<li>Prepare the mouse
	<ol>
		<li>Place the mouse in a supine position in a glass dish using blunt forceps, or pin it to a dissection board.</li>
		<li>Add a thin layer of pre-warmed PBS to a Petri dish.</li>
		<li>Change to a fresh pair of gloves.</li>
		<li>Lift the lower abdominal skin with forceps and make a small horizontal incision using scissors to open the abdominal cavity.</li>
		<li>Make a vertical incision (~2 cm from caudal to rostral) extending from the initial cut to expose the internal organs.</li>
	</ol>
	</li>
	<li>Isolate the fat pad
	<ol>
		<li>Using small sharp scissors and pointed forceps, locate and excise the white adipose tissue. Use a separate set of instruments to prevent contamination.</li>
		<li>Male Mice: Gonadal fat is attached only to the epididymis and testis. Carefully lift the fat pad and cut above the epididymis, or excise the epididymis and testis together with the gonadal fat, and remove them afterward.</li>
		<li>Female mice: Gonadal fat is attached to the uterine horn and ovaries and is more prominent than in males. Carefully lift the fat pad and excise along the uterine horn and ovaries.</li>
		<li>Place the isolated fat pads into the PBS-filled Petri dish.</li>
		<li>Remove residual epididymis, testis, ovaries, or blood vessels using a razor blade.</li>
		<li>Transfer the cleaned fat pad into a second Petri dish containing pre-warmed PBS. Optionally weigh the fat pads. 
		NOTE: Weighing the fat pads before culture allows normalization of tissue mass between experimental conditions.</li>
	</ol>
	</li>
</ol>
<img alt="Mouse necropsy process, organ extraction, tissue preparation, cell culture setup for biological analysis." class="xfigimg" src="/files/ftp_upload/71651/71651fig2v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Isolation and culture of visceral adipose tissue explants. (A,B) Opening the abdominal cavity and exposing visceral adipose tissue. (C, D) Isolation of epididymal adipose tissue from male mice, fat is attached to the epididymis (red circles) and the testis (blue circles). (E, F) Isolation of gonadal adipose tissue from female mice, fat is attached to the uterine horn (green circles) and ovaries. (G, H) Preparation and trimming of adipose tissue explants. (I, J) Transfer of explants into culture wells and placement of cell culture inserts. (K, L) Representative images of explants during culture in 6-well plates. <a href="https://www.jove.com/files/ftp_upload/71651/71651fig2largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Explant preparation and culture
<ol>
	<li>Prepare explants 
	CAUTION: Razor blades are sharp and may cause injury. Handle with care and dispose of blades in accordance with institutional safety procedures.
	<ol>
		<li>Cut the fat pad into strips and then into cubes of approximately 10 mm3 (~10 mg per explant) using a razor blade.</li>
	</ol>
	</li>
	<li>Establish explant cultures
	<ol>
		<li>Add 1–1.5 mL of culture medium to each well of a 6-well plate. 
		NOTE: For normalization, use approximately 50 mg of adipose tissue per 1 mL of culture medium.</li>
		<li>Transfer five explants into each well using a sterile spoon or spatula.</li>
		<li>Arrange the explants evenly in a circular pattern.</li>
		<li>Cover the explants with a cell culture insert using sterile forceps. 
		NOTE: Avoid introducing air bubbles when positioning the inserts, and ensure the explants are centered within them.</li>
		<li>Incubate the explants at 37 °C, 5% CO₂, and 21% O₂ for 7–10 days. 
		NOTE: Use pre-warmed PBS and culture medium to enhance tissue viability. Work quickly to minimize the time that fat pads remain outside the incubator. Avoid contamination with mouse fur. If hair adheres to the tissue, remove it and rinse the tissue with warm PBS.</li>
	</ol>
	</li>
</ol>
4. Downstream analyses
<ol>
	<li>After the 7–10 day culture period, collect explants, stromal vascular fraction cells, or culture supernatants for downstream analyses, including microscopy, flow cytometry, qPCR, ELISA, or RNA sequencing, as required for the experimental endpoint.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform statistical analyses using GraphPad Prism 10.2. Screen data sets for outliers using the ROUT test (Q = 1%) and assess normality using the Shapiro–Wilk test (α = 0.05).</li>
	<li>Compare two groups using paired Student's t-tests or Wilcoxon matched-pairs tests, as appropriate. Compare multiple groups using one-way ANOVA followed by Dunnett's post hoc test. Consider differences statistically significant at p &lt; 0.05.</li>
	<li>Present data as mean ± SEM. Unless otherwise indicated, define n as the number of biological replicates (individual mice).</li>
</ol>

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	<li>Braune J, et al. Multinucleated giant cells in adipose tissue are specialized in adipocyte degradation. Diabetes. 2021;70(2):538-548. doi:10.2337/db20-0293.</li>
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	<li>Gericke MT, et al. Receptors for NPY and PACAP differ in expression and activity during adipogenesis in the murine 3T3-L1 fibroblast cell line. Br J Pharmacol. 2009;157(4):620-632. doi:10.1111/j.1476-5381.2009.00164.x.</li>
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The authors declare no competing interests.

Given the increasing global prevalence of metabolic diseases and diabetes, understanding the mechanisms underlying adipose tissue (AT) inflammation remains an important research objective. To address this need, an ex vivo explant culture system that enables the induction and analysis of AT inflammation under controlled experimental conditions was developed .
A critical requirement of this method is the preservation of tissue viability throughout the culture period. Using multiple comp...

Adipose tissue (AT) is a metabolically active organ composed of approximately 20% adipocytes and a heterogeneous mixture of immune cells, fibroblasts, progenitor cells, and vascular cells in humans1. AT is classified into unilocular white adipose tissue (WAT), multilocular brown adipose tissue (BAT), and intermediate beige adipose tissue, reflecting heterogeneity in composition, function, and anatomical location. WAT is distributed among subcutaneous (SCAT), visceral (VAT), perivascular, and marrow adipose depots, which differ in developmental origin, gene expression profiles, and metabolic function. Beyond its role in lipid storage, AT functions as an endocrine organ by secreting cytokines, hormones, and adipokines2. Compared with SCAT, VAT is more metabolically active and enriched in inflammatory cells, making it particularly relevant for studies of AT inflammation and metabolic diseases such as type 2 diabetes3,4,5.
AT is a key regulator of metabolic health and plays a central role in obesity, which is associated with numerous comorbidities. Obesity promotes adipocyte hypertrophy, hyperplasia, and proliferation6, while AT expansion and inflammation strongly correlate with hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and impaired glucose tolerance3. AT expansion is presumed to be accompanied by increased adipocyte death, with approximately 10% cellular turnover annually7, triggering macrophage recruitment that is essential for tissue clearance and remodeling8. Because adipocytes are substantially larger (100–200 µm) than macrophages, efficient phagocytosis by these approximately fivefold smaller cells is challenging, thereby impairing tissue homeostasis and promoting chronic AT inflammation9. Adipose tissue macrophages (ATMs) mediate the clearance of oversized dying adipocytes either by fusing into multinucleated giant cells (MGCs) or by forming crown-like structures (CLS) around dead adipocytes and degrading them through lysosomal exocytosis10,11,12. CLS abundance correlates with obesity-associated inflammation11,13.
In healthy AT, ATMs are predominantly anti-inflammatory M2-like macrophages, whereas obesity promotes a shift toward a pro-inflammatory M1-like phenotype14,15. Additional mechanistically distinct macrophage subsets emerge, including metabolically activated macrophages (MMes) and lipid-associated macrophages (LAMs), which play important roles in the clearance of dead adipocytes through lysosomal exocytosis16,17,18.
To investigate AT inflammation in a physiologically relevant multicellular environment, the protocol established an ex vivo mouse model based on organotypic explant cultures of VAT derived from lean mice. VAT explants cultured for 7–10 days develop an inflammatory phenotype comparable to that observed after at least 12 weeks of high-fat diet (HFD) feeding with respect to CLS formation, macrophage phenotype, and inflammatory cytokine production9,19. Importantly, this approach reduces animal use, eliminates the need for dietary intervention approvals, and contributes to the 3R principles by reducing the reliance on long-term dietary intervention studies. Compared with conventional two-dimensional (2D) cell culture systems, such as single-cell culture models (e.g., 3T3-L1 cells or primary adipocyte cultures), this method preserves tissue complexity and cellular diversity, thereby enabling the investigation of physiological cell–cell interactions, including those between immune cells and adipocytes.
Cultured AT explants are compatible with a wide range of analytical approaches. Live confocal imaging enables the monitoring of ATM accumulation, CLS formation, and MGC formation9,19. Collagenase digestion allows isolation of the stromal vascular fraction (SVF) for flow cytometric analyses, including proliferation assays and determination of M1-like, M2-like, or MMe phenotypes12. Gene expression analyses, including quantitative PCR (qPCR) and RNA sequencing, can be performed on whole explants, SVF cells, or sorted macrophages9. Protein expression and enzymatic activity can be assessed by Western blotting or ELISA12,20,21, while tissue architecture and marker expression can be visualized by whole-mount staining and immunofluorescence9,22. In addition, pharmacological and genetic interventions, including ATM depletion and cytokine treatment, are readily feasible21,23,24.
In summary, this ex vivo mouse model of HFD-like AT inflammation enables investigation of adipocyte death, direct in situ observation of cellular behavior, and pharmacological or genetic manipulations relevant to AT immunology (Figure 1).
<img alt="Adipose tissue culture process; diagram showing extraction, cutting, incubation, and inflammation." class="xfigimg" src="/files/ftp_upload/71651/71651fig1v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Graphical summary of the workflow. Visceral adipose tissue (AT) is isolated from mice, cut into small explants, and cultured for 7–10 days. During the culture period, explants develop an inflammatory phenotype, enabling the study of adipose tissue inflammation under controlled ex vivo conditions. <a href="https://www.jove.com/files/ftp_upload/71651/71651fig1largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>BD OptEIA Mouse TNF ELISA Kit</td><td></td><td>BD Biosciences</td><td>560478</td></tr><tr><td>Bodipy 558/568 C12</td><td>Flow cytometry: 1:10000; Staining: 1:2000</td><td>Invitrogen</td><td>D3835</td></tr><tr><td>Bovine Serum Albumin</td><td>3%</td><td>Sigma</td><td>A9647</td></tr><tr><td>CD107a (LAMP1) &amp;ndash; AF647</td><td>Staining: 1:100; Flow cytometry: 1:200</td><td>BioLegend</td><td>121610</td></tr><tr><td>CD11c &amp;ndash; BV605</td><td>Flow cytometry: 1:100</td><td>BioLegend</td><td>117334</td></tr><tr><td>CD16/32</td><td>Flow cytometry: 1:100</td><td>Invitrogen</td><td>14-0161-82</td></tr><tr><td>CD301 &amp;ndash; AF647</td><td>Flow cytometry: 1:50</td><td>Bio-Rad</td><td>MCA2392A647</td></tr><tr><td>CD36 &amp;ndash; PE</td><td>Flow cytometry: 1:400</td><td>BioLegend</td><td>102606</td></tr><tr><td>CD45 &amp;ndash; PE</td><td>Flow cytometry: 1:100</td><td>BD Pharmingen</td><td>555483</td></tr><tr><td>Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit</td><td>EdU: 5 &amp;micro;M</td><td>Invitrogen</td><td>C10632</td></tr><tr><td>Clodronate Liposomes</td><td>500 &amp;micro;g/ml clodronate</td><td>Liposoma</td><td></td></tr><tr><td>Collagenase Type 2</td><td>1 mg/ml, 315 U/mg</td><td>Worthington</td><td>LS004176</td></tr><tr><td>CyQUANT LDH Cytotoxicity Assay Kit</td><td></td><td>Invitrogen</td><td>C20301</td></tr><tr><td>DAPI</td><td>Flow cytometry: 0.2 &amp;micro;g/ml</td><td>Thermo Fisher</td><td>62248</td></tr><tr><td>Ethanol</td><td>70%</td><td>Carl Roth</td><td>T913.1</td></tr><tr><td>F4/80 &amp;ndash; AF647</td><td>Flow cytometry: 1:100</td><td>Invitrogen</td><td>MF48021</td></tr><tr><td>F4/80 &amp;ndash; PE-Cy7</td><td>Flow cytometry: 1:200</td><td>Invitrogen</td><td>25-4801-82</td></tr><tr><td>Fetal Calf Serum</td><td>1.10</td><td>Gibco</td><td>10270106</td></tr><tr><td>Fetal Calf Serum</td><td>1.20</td><td>Gibco</td><td>10270106</td></tr><tr><td>HBSS (-Mg, -Ca)</td><td></td><td>Gibco</td><td>14170138</td></tr><tr><td>HEPES</td><td>13 mM</td><td>Roth</td><td>HN77.2</td></tr><tr><td>Hoechst 33342</td><td>1:10,000</td><td>Life Technologies</td><td>H3570</td></tr><tr><td>LysoTracker &amp;ndash; AF647</td><td>Flow cytometry: 1:20000; Staining: 1:10000</td><td>Invitrogen</td><td>H34477</td></tr><tr><td>MAC-2</td><td>Staining: 1:1000</td><td>Cedarlane</td><td>CL8942AP</td></tr><tr><td>Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 &amp;micro;m</td><td></td><td>Merck Millipore</td><td>PICM0RG50</td></tr><tr><td>PBS (-Mg, -Ca)</td><td></td><td>Gibco</td><td>14190169</td></tr><tr><td>PBS (-Mg, -Ca)</td><td></td><td>Gibco</td><td>14190169</td></tr><tr><td>Penicillin/Streptomycin (10,000 U/ml)</td><td>0.111111111</td><td>Gibco</td><td>11548876</td></tr><tr><td>Penicillin/Streptomycin (10,000 U/ml)</td><td>0.111111111</td><td>Gibco</td><td>11548876</td></tr><tr><td>Perilipin</td><td>2 &amp;micro;g/ml</td><td>Abcam</td><td>ab3526</td></tr><tr><td>Plexxicon 5622</td><td>40 &amp;micro;M</td><td>Plexxicon Inc.</td><td>HY-114153</td></tr><tr><td>RPMI-Media 1640</td><td>with 11 mM glucose</td><td>Sigma Aldrich</td><td>R8758</td></tr><tr><td>RPMI-Media 1640</td><td></td><td>Sigma</td><td>R8758</td></tr><tr><td>TC-Insert for 6-well plates</td><td></td><td>Sarstedt</td><td>83.3930.040</td></tr><tr><td>TPP tissue culture test plate 6</td><td></td><td>TPP</td><td>92006</td></tr><tr><td>Triton X-100</td><td>1%</td><td>Carl Roth</td><td>3051.2</td></tr><tr><td>Zinc chloride</td><td>0.8 mM</td><td>Sigma</td><td>Z0152</td></tr><tr><td>Zinc formaline</td><td></td><td>Polyscience</td><td>21516-3.75</td></tr></tbody></table>

an ex vivo mouse model for high-fat diet-like adipose tissue inflammation

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

This protocol describes an ex vivo mouse model that recapitulates selected cellular and inflammatory features observed in visceral adipose tissue (VAT) following high-fat diet (HFD) feeding, without dietary intervention in vivo. Small VAT explants from lean mice are cultured for 7&#8211;10 days, during which they spontaneously develop an inflammatory phenotype that closely resembles that of VAT from HFD-fed animals. Importantly, the model preserves adipose tissue (AT) architecture, enabling the investigation of inflammatory processes in a physiologically relevant context. During culture, key hallmarks of obesity-related inflammation emerge, including the formation of crown-like structures (CLS), the accumulation of metabolically activated macrophages (MMes), and increased inflammatory cytokine production. Despite adipocyte death, other drivers of obesity-related inflammation, including metabolic stressors such as saturated fatty acids, altered lipid handling, glucose excess, oxidative stress, hypoxia, adipokine imbalance, and paracrine adipocyte&#8211;immune cell signaling, can be partially represented in the model. The system supports a wide range of downstream applications, including live imaging, flow cytometry, quantitative PCR, ELISA, immunostaining, and bulk or single-cell RNA sequencing. In addition, pharmacological and cytokine-based treatments can be applied to investigate signaling pathways and cell type-specific responses. This easily customizable model aligns with the 3R principles by reducing animal use for in vivo dietary studies. The model is reproducible, cost-effective, and straightforward to implement, providing a versatile platform for studying adipocyte death, immune cell dynamics, and therapeutic strategies in AT inflammation.

General tissue viability was maintained throughout the culture period and was monitored using several complementary approaches. Tissue viability can be assessed by confocal microscopy using viability dyes such as Calcein25, antibody staining against Perilipin to evaluate adipocyte membrane integrity17,26, or transgenic reporter protein expression in adipocytes. Tissue vitality and integrity were preserved for up to 14 days in culture, alth...

Using an ex vivo explant model, small pieces of the rostral portion of visceral white adipose tissue from lean mice are cultured for 7&#8211;10 days, enabling the induction and study of adipose tissue inflammation as it occurs during high-fat diet feeding.

This protocol describes an ex vivo mouse model that recapitulates selected cellular and inflammatory features observed in visceral adipose tissue (VAT) ...

We are investigating inflammation in adipose tissue, focusing on the role of adipose tissue macrophages in this process. This model can be applied in a great variety of adipose tissue inflammation research where the focus is on the adipose tissue itself. To begin, prepare the culture medium under sterile conditions.Warm the culture medium and sterile PBS to 37 degrees Celsius. Clean all equipment and the work area inside the tissue culture hood with 70%ethanol. After placing the sacrificed animal in a glass beaker containing 70%ethanol, wipe the exterior of the glass beaker with ethanol and transfer it into the tissue culture hood.Using blunt forceps, place the mouse in a supine position, and pin it to a dissection board. Then, add a thin layer of pre-warmed PBS to a Petri dish. Change to a fresh pair of gloves.Using forceps, lift the lower abdominal skin, and make a small horizontal incision using scissors to open the abdominal cavity. Make a vertical incision extending from the initial cut to expose the internal organs. For male mice, carefully lift the gonadal fat pad, which is attached to the epididymis and testis.Make a cut above the epididymis. Alternatively, excise the epididymis and testis together with the gonadal fat, and remove the epididymis and testis afterward. For female mice, carefully lift the gonadal fat pad, which is attached to the uterine horn and ovaries, and excise it along the uterine horn and ovaries.Place the isolated fat pads into a Petri dish filled with PBS. Using a razor blade, remove any residual epididymis, testis, ovaries, or blood vessels from the fat pads. Transfer the cleaned fat pad into a second Petri dish containing pre-warmed PBS.Using a razor blade, cut the fat pad into strips, and then cut the strips into cubes of approximately 10 cubic millimeters. Add one to 1.5 milliliters of culture medium to each well of a six-well plate. Using a sterile spoon or spatula, transfer five explants into each well.Arrange the explants evenly in a circular pattern. Using sterile forceps, cover the explants with a cell culture insert. Incubate them at 37 degrees Celsius, 5%carbon dioxide, and 21%oxygen for seven to 10 days.After a culture period of seven to 10 days, collect explants or culture supernatants for downstream analyses as required for the experimental endpoint. TD tomato expression remains stable throughout the 14-day culture period. Lactate dehydrogenase levels remained low, relative to maximal release following complete tissue lysis, indicating preserved tissue integrity.Perilipin-positive adipocytes remained intact for up to 10 days of culture. The proportion of DAPI positive cells remained stable throughout the culture period. Expression of PPARG, AdipoQ, LEP, and LPL remained stable throughout the culture period.A key feature of adipose tissue inflammation is the formation of crown-like structures by adipose tissue macrophages around dying adipocytes, primarily occurring between days five and seven of culture. Expression of inflammatory genes and tumor necrosis factor alpha increased over time. Multinucleated giant cells developed and contributed to dead adipocyte clearance.Although macrophages accumulated within crown-like structures and fused into multinucleated giant cells, the overall number of adipose tissue macrophages remained relatively stable, as a proliferation peak was observed on day five. The adipose tissue macrophages phenotype shifted from a predominantly M2-like state at day one toward an increased proportion of M1-like macrophages at day seven. Metabolically activated macrophages increased during culture with increased body piece staining and increased lysosomal content.Multiple macrophage populations were identified, including pro-inflammatory, metabolically activated, proliferating, inflammation-resolving, and anti-inflammatory macrophages. Clodrenate liposomes reduced macrophage numbers by approximately 60%by day seven. And PLX-5622 reduced macrophage numbers by approximately 70%by day seven.This method can be used to study the intercellular functional processes and alterations that occur during adipose tissue inflammation. The most important challenge when performing this protocol is to handle the tissue as gently as possible, so that the tissue explants remain viable throughout the culture period. Future studies can use this method to dissect adipose tissue inflammation signaling, evaluate cell-specific responses to therapies and reduce animal use.

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Research

JoVE Journal

Immunology and Infection

Adipose tissue (AT) is a site of intense immune cell activation and interaction.  Almost all cells of the immune system are present in AT and their ratios are altered by obesity.  Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolation of Adipose Tissue Immune Cells

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

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

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Leipzig University.  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

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