Name: Caracterização da secreção de vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP In Vivo e In Vitro (Vídeo) | JoVE Journal
Uploaded: 2025-12-05T13:20:00.000+00:00
Duration: 10 min 40 s
Description: Aprenda a caracterizar vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP in vivo e in vitro. Este método permite visualizar e quantificar a secreção de vesículas no tecido adiposo.

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</ol>

<table><tbody><tr><td>0,5 M EDTA, pH 8,0</td><td>Fisher</td><td>AM9260G</td><td>Diluição 500x</td></tr><tr><td>10x PBS</td><td>Fisher Scientific</td><td>70-013-032</td><td>1000x diluição com água</td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td>1000x diluição</td></tr><tr><td>Kit de Isolamento de Progenitores do Tecido Adiposo, camundongo</td><td>Miltenyi biotec</td><td>30-106-639</td><td>Pronto para uso</td></tr><tr><td>Filtro Centrífugo Amicon Ultra, MWCO de 100 kDa</td><td>Sigma</td><td>UFC9100</td><td>Volume de amostra de 15 mL</td></tr><tr><td>Filtro Centrífugo Amicon Ultra, MWCO de 100 kDa</td><td>Sigma</td><td>UFC9100</td><td>Volume de amostra de 4 mL</td></tr><tr><td>BSA</td><td>Sigma</td><td>A6003</td><td>Pronto para uso</td></tr><tr><td>Anticorpo de Calnexina</td><td>Calnexina</td><td>AB22595</td><td>1000x diluição</td></tr><tr><td>Anticorpo CD63</td><td>Abcam</td><td>ab217345</td><td>Diluição 500x</td></tr><tr><td>CD81</td><td>Tecnologia de sinalização celular</td><td>10037s</td><td>Diluição 500x</td></tr><tr><td>Corning&nbsp; BioCoat&nbsp; Slide da Cultura Colágeno I</td><td>Sigma</td><td>CLS354630</td><td>Pronto para uso</td></tr><tr><td>Corning&nbsp; Filtro de células</td><td>Sigma</td><td>CLS431750</td><td>40 & mu; m tamanho dos poros</td></tr><tr><td>Corning&nbsp; Filtro de células</td><td>Sigma</td><td>CLS431752</td><td>100 & mu; m tamanho dos poros</td></tr><tr><td>Anticorpo COX IV</td><td>Tecnologia de Sinalização Celular</td><td>4844</td><td>1000x diluição</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td>Pronto para uso</td></tr><tr><td>DMEM/F-12, suplemento GlutaMAX</td><td>Thermofisher</td><td>10565018</td><td>Pronto para uso</td></tr><tr><td>Dnase 1</td><td>Sigma</td><td>D4513</td><td>Estoque 5 KU/mL em -20 &deg; C</td></tr><tr><td>Suplemento GlutaMAX</td><td>Gibco</td><td>35050061</td><td>100x diluição</td></tr><tr><td>Halt & trade; Cocktail de Protease e Inibidor de Fosfatase (100X)</td><td>Thermofisher</td><td>78444</td><td>Pronto para uso</td></tr><tr><td>Anticorpo HSP70</td><td>Abcam</td><td>ab181606</td><td>1000x diluição</td></tr><tr><td>Invitrogen eBioscience 10X Lysis Buffer de RBC (Multiespécies)</td><td>Fisher Scientific</td><td>50-112-9743</td><td>10x diluição em água</td></tr><tr><td>Izon Science Usa Ltd QEVORIGINAL 70NMGEN2 5/PK SDP</td><td>Fisher Scientific</td><td>NC2042225</td><td>Pronto para uso</td></tr><tr><td>Microscópio confocal LEICA STELLARIS 8</td><td>Microscópios Leica</td><td>N/A</td><td></td></tr><tr><td>Liberase TM Grau de Pesquisa</td><td>Sigma</td><td>LIBTM-RO</td><td>Estoque 5 mg/mL a -20 °C; C</td></tr><tr><td>Filtro de seringa Millex MCE</td><td>Sigma</td><td>SLAA025</td><td>0,8 & mu; m tamanho dos poros</td></tr><tr><td>NanoSight Pro</td><td>Malvern Panalytical</td><td>N/A</td><td></td></tr><tr><td>Novex Tris-Glicina Mini Géis de Proteína, 4&ndash; 12%,</td><td>Thermofisher</td><td>XP04120BOX</td><td>Pronto para uso</td></tr><tr><td>Buffer de Amostra SDS Tris-Glicina Novex (2x)</td><td>Thermofisher</td><td>LC2676</td><td>Pronto para uso</td></tr><tr><td>Anticorpo anti-camundongo Ly-6A/E (Sca-1) Azul do Pacífico</td><td>BioLegend</td><td>108119</td><td>Diluição 25x</td></tr><tr><td>Solução de paraformaldeído (16%)</td><td>Fisher Scientific</td><td>50-980-487</td><td>diluir para 4% com PBS</td></tr><tr><td>pluriStrainer S / 30 & micro; m (Filtro de células)</td><td>Fisher Scientific</td><td>NC0922459</td><td>30 & mu; m tamanho dos poros</td></tr><tr><td>Solução Ponceau S</td><td>Sigma</td><td>P7170-1L</td><td>Pronto para uso</td></tr><tr><td>Primocin</td><td>Invivogen</td><td>NC9141851</td><td>Diluição 500x</td></tr><tr><td>Reagente Antifade de Ouro ProLong com DAPI</td><td>Tecnologia de Sinalização Celular</td><td>8961S</td><td>Pronto para uso</td></tr><tr><td>Anticorpo anti-camundongo purificado CD16/32</td><td>BioLegend</td><td>101301</td><td>100x diluição</td></tr><tr><td>Buffer RIPA&nbsp;</td><td>Fisher Scientific</td><td>PI89900</td><td>10x diluição em água</td></tr><tr><td>Citômetro de fluxo espectral (Aurora 3)</td><td>Cytek Biosciences</td><td>N/A</td><td></td></tr><tr><td>Anticorpo Sintaxina 4</td><td>Sistemas Sinápticos</td><td>110042</td><td>Diluição 5000x</td></tr><tr><td>Tecnai 20 (Microscópio Eletrônico de Transmissão)</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>Anticorpo vinculina</td><td>Abcam</td><td>ab18508</td><td>Diluição 5000x</td></tr></tbody></table>

caracterização da secreção de vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter cd63-gfp in vivo e in vitro

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

Usando esse protocolo de detecção de ADEVs por meio da captação de APCs por microscopia confocal, isolamos APCs dos tecidos adiposos inguinais de camundongos AdipCD63-GFP e Stopfl/fl/CD63-GFP e os colorimos com DAPI para visualização dos núcleos. Quando examinados por microscopia confocal, APCs de camundongos Stopfl/fl/CD63-GFP não mostraram pontos de GFP detectáveis (Figura 1A). Em contraste, APCs isolados de camundongos AdipCD63-GFP apresentaram numerosos pontos disti...

Caracterização da secreção de vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP In Vivo e In Vitro

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

characterization-adipocyte-derived-extracellular-vesicle-secretion

Research

JoVE Journal

Biology

Biologia

A camada de superfície endotelial glicocálice / endotelial está idealmente estudada por microscopia intravital. Microscopia intravital é tecnicamente desafiante em um órgão em movimento, tais como o pulmão. Demonstramos como brightfield simultânea e microscopia de fluorescência pode ser utilizado para estimar a espessura da camada endotelial da superfície em um movimento...

Na medição in vivo da camada superficial do rato pulmonar endotelial

Este protocolo descreve métodos para purificar, quantificar e caracterizar vesículas extracelulares (EVs) / exossomos de células epiteliais mamárias não aderentes / mesenquimatosas e para utilizá-las para transferir a capacidade de formação de glândula mamária para células epiteliais mamárias luminal. EVs / exosomas derivados de células epiteliais mamárias semelhantes a tronco podem transferir esta propriedade celular para células que ingerem EVs / exossos.

Transferência de capacidade de formação de glândulas mamárias entre células epiteliais basais mamárias e células luminal mamárias através de vesículas / exossos extracelulares

A diferenciação dos adipócitos brancos e bege de tecido adiposo vascular progenitores tem potencial para melhoria metabólica em obesidade. Descrevemos os protocolos para um CD34 + CD31 + isolamento de células endoteliais de gordura humana e uma subsequente em vitro expansão e diferenciação em adipócitos brancos e bege. Diversas aplicações a jusante são discutidas.

Isolamento, expansão e indução de Adipogenic de CD34 + CD31 + células endoteliais de tecido adiposo humano de Omental e subcutâneo

Neste protocolo, descrevemos o fluxo de trabalho completo para isolamento rápido das vesículas extracelulares de sangue total humano e caracterização de marcadores específicos pela análise de controle de nanopartículas baseada em fluorescência. Os resultados apresentados mostram um elevado nível de reprodutibilidade e podem ser ajustados para sobrenadantes de cultura de células.

Caracterização de baseada em fluorescência rápida das vesículas extracelulares única no sangue humano, com análise de acompanhamento de nanopartículas

Descrevemos um sistema de cultura in vitro de matriz extracelular em 3D que permite a dissecação dos papéis da matriz e dos adipócitos em contribuir para o fenótipo metabólico do tecido adiposo.

Um modelo de cultura de matriz-adipócito extracelular humano para estudar crosstalk metabólico de matriz e células

A última geração de ferramentas de caracterização de EV é capaz de uma única análise de EV em vários parâmetros simultaneamente. A citometria de nanofluxo mede todas as partículas biológicas maiores que 45 nm sem marcação e identifica características específicas de subpopulações por uma variedade de técnicas de marcação fluorescente.

Caracterização da Proteína Transmembrana da Vesícula Extracelular Única por Citometria de Nanofluxo

O presente protocolo descreve a centrifugação diferencial para isolar e caracterizar EVs representativos (exossomos e microvesículas) de CTMs humanas cultivadas. Outras aplicações desses EVs também são explicadas neste artigo.

Isolamento, Caracterização e Aplicação Terapêutica de Vesículas Extracelulares a partir de Células-Tronco Mesenquimais Humanas Cultivadas

Este protocolo descreve todos os procedimentos, desde a cultura de células-tronco mesenquimais derivadas do tecido adiposo humano (ADSCs) e coleta de sobrenadante até a extração de vesículas extracelulares (EVs) usando ultracentrifugação.

Purificação e Caracterização de Vesículas Extracelulares de Células-Tronco Mesenquimais Derivadas de Tecido Adiposo Humano

Aqui, descrevemos um protocolo para isolar, cultivar e fenotipar células endoteliais microvasculares do tecido adiposo subcutâneo humano (hSATMVECs). Além disso, descrevemos um modelo experimental de diafonia hSATMVEC-adipócitos.

Estudando a conversa cruzada de células endoteliais adiposas/adipócitos no tecido adiposo subcutâneo humano

Usando um modelo explantado ex vivo , pequenos pedaços da porção rostral do tecido adiposo branco visceral de camundongos magros são cultivados por 7 a 10 dias, permitindo a indução e o estudo da inflamação do tecido adiposo conforme ocorre durante a alimentação rica em gordura.

Um modelo de camundongo ex vivo para inflamação do tecido adiposo com dieta rica em gordura

Aprenda a caracterizar vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP in vivo e in vitro. Este método permite visualizar e quantificar a secreção de vesículas no tecido adiposo.

Caracterização da secreção de vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP In Vivo e In Vitro (Vídeo) | JoVE Journal

Caracterização da secreção de vesículas extracelulares derivadas de adipócitos usando um modelo de camundongo repórter CD63-GFP In Vivo e In Vitro

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