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<ol>
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```html
<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) &ndash; AF647</td><td>Staining: 1:100; Flow cytometry: 1:200</td><td>BioLegend</td><td>121610</td></tr><tr><td>CD11c &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 &ndash; AF647</td><td>Flow cytometry: 1:50</td><td>Bio-Rad</td><td>MCA2392A647</td></tr><tr><td>CD36 &ndash; PE</td><td>Flow cytometry: 1:400</td><td>BioLegend</td><td>102606</td></tr><tr><td>CD45 &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 &micro;M</td><td>Invitrogen</td><td>C10632</td></tr><tr><td>Clodronate Liposomes</td><td>500 &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 &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 &ndash; AF647</td><td>Flow cytometry: 1:100</td><td>Invitrogen</td><td>MF48021</td></tr><tr><td>F4/80 &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 &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 &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 &micro;g/ml</td><td>Abcam</td><td>ab3526</td></tr><tr><td>Plexxicon 5622</td><td>40 &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>
```

un modello topino ex vivo per l'infiammazione del tessuto adiposo ad alto contenuto di grassi

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

Un modello topino ex vivo per l'infiammazione del tessuto adiposo ad alto contenuto di grassi

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.

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

Research

Ricerca

JoVE Journal

Immunology and Infection

Immunologia e Infezioni

Il tessuto adiposo (AT) è un sito di attivazione delle cellule immunitarie intensa e interazione. Quasi tutte le cellule del sistema immunitario sono presenti in AT e per i rapporti sono alterati da obesità. Isolamento adeguato, quantificazione e caratterizzazione di AT popolazioni di cellule immunitarie sono fondamentali per comprendere il loro ruolo nella malattia immunometabolic.

Isolamento di tessuto adiposo cellule immunitarie

Il tessuto adiposo (AT) può influenzare l'omeostasi di tutto il corpo, quindi è importante comprendere i meccanismi molecolari della differenziazione e della funzione degli adipociti. Forniamo un protocollo per ottenere nuove informazioni su questi processi analizzando l'omeostasi degli adipociti, la differenziazione e l'esposizione all'ipossia come modello per l'apoptosi indotta degli adipociti.

Meccanismo di regolazione dei numeri adipociti in organismi adulti attraverso la differenziazione e l&#39;apoptosi omeostasi

È descritto un semplice e affidabile modello animale di roditore indotto dalla dieta per la steatoepatite non alcolica (NASH), ottenuto attraverso l'alloggiamento non SPF degli animali e la somministrazione di una dieta specifica ad alto contenuto di grassi. Descriviamo l'identificazione di sottoinsiemi di cellule immunitarie epatiche e adiposo per ricapitolare le condizioni immunologiche umane esponendo i topi ai germi ambientali.

Un modello avanzato di murine per la steatoepatite non alcolica in associazione con il diabete di tipo 2

Descriviamo un sistema 3D di coltura extracellulare extracellulare umana-adipocito in vitro che permette la dissezione dei ruoli della matrice e adipociti nel contribuire al fenotipo metabolico del tessuto adiposo.

Un modello di coltura a matrice extracellulare extracellulare -adipocyte umano per lo studio del crosstalk metabolico a matrice

Questo protocollo descrive l'approccio tecnico per isolare sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi di tessuto adiposo bianco (WAT) murino intra-addominale mediante smistamento cellulare attivato dalla fluorescenza o separazione di microsfere immunomagnetiche.

Isolamento di sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi adiposi intra-addominali murini

Questo protocollo descrive l'isolamento dei preadipociti di topo dal grasso sottocutaneo, la loro differenziazione in adipociti maturi e l'induzione della resistenza all'insulina. L'azione dell'insulina viene valutata mediante fosforilazione/attivazione dei membri della via di segnalazione dell'insulina attraverso il western blot. Questo metodo consente la determinazione diretta della resistenza/sensibilità all'insulina negli adipociti primari.

Adipociti di topo differenziati in coltura primaria: un modello di insulino-resistenza

La lipolisi dei trigliceridi negli adipociti è un importante processo metabolico che porta alla liberazione degli acidi grassi liberi e del glicerolo. Qui, forniamo un protocollo dettagliato per misurare la lipolisi basale e stimolata negli adipociti e nel tessuto adiposo ex vivo dei topi.

Misurazione del tasso di lipolisi nel tessuto adiposo murino ex vivo e nei preadipociti primari differenziati in vitro

Questo protocollo descrive l'isolamento semi-automatico della frazione vascolare stromale (SVF) dal tessuto adiposo murino per ottenere preadipociti e ottenere la differenziazione degli adipociti in vitro. L'uso di un dissociatore tissutale per la digestione della collagenasi riduce la variazione sperimentale e aumenta la riproducibilità.

Isolamento semi-automatico della frazione vascolare stromale dal tessuto adiposo bianco murino mediante un dissociatore tissutale

Questo protocollo presenta un approccio dettagliato alla replicazione di un modello murino di insufficienza cardiaca indotta da iperlipidemia con frazione di eiezione conservata (HFpEF). Il disegno combina la somministrazione del virus adeno-associato 9-troponina cardiaca T-recettore delle lipoproteine a bassa densità (AAV9-cTnT-LDLR) e del poloxamer-407 (P-407).

Un modello murino di insufficienza cardiaca indotta da iperlipidemia con frazione di eiezione conservata

Schistosoma mansoni uova sono potenti stimolatori del T helper di tipo 2 (Th2) risposta immunitaria, caratteristica di infezioni parassitarie, asma e infiammazione allergica. Questo protocollo utilizza S. mansoni uovo iniezione per generare un CD4 Th2 citochine indotta da risposta infiammatoria nel polmone, caratterizzata dalla formazione di granulomi intorno al polmone l'uovo, eosinofilia e l'attivazione dei macrofagi alternative.

Con ovuli<em&gt; Schistosoma mansoni</em&gt; Come<em&gt; In vivo</em&gt; Modello di elminti-indotta infiammazione polmonare

Leipzig University.  Utilizzando un modello ex vivo di espianti, piccoli pezzi della porzione rostrale del tessuto adiposo bianco viscerale di topi magri vengono coltivati per 7–10 giorni, permettendo l'induzione e lo studio dell'infiammazione del tessuto adiposo durante l'alimentazione ricca di grassi.

Un modello topino ex vivo per l'infiammazione del tessuto adiposo ad alto contenuto di grassi

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