Reconhecemos a Instalação de Citometria do Núcleo de Análise celular da Clínica Mayo clinic para assistência com a triagem FACS.

Todos os procedimentos experimentais em animais foram realizados sob aprovação do Comitê Institucional de Cuidados e Uso de Animais da Clínica Mayo.
1. Preparação da solução 
<ol><li>Prepare a solução de colagenase 2% (w/v) dissolvendo a colagemnase II 2 g na solução de sal balanceada (HBSS) de 100 mL da Hanks. Alíquota de 200 μL cada para cada uso.</li>
	<li>Prepare o meio de neutralização misturando 84 mL F-12 médio, 15 mL de serum de cavalo e penicilina/estreptomicina de 1 mL.</li>
	<li>Prepare o tampão de citometria de fluxo dissolvendo 500 mg de albumina de soro bovino (BSA) e 400 μL de 0,5 M EDTA em 100 mL da linha salina tamponada de fosfato de Dulbecco (DPBS).</li>
</ol>2. Dissecção da almofada de gordura epidídica e dissociação tecidual
<ol><li>Eutanize humanamente (luxação isoflurane/cervical) de um rato macho. Neste protocolo, foi usado um rato FVB de quatro meses de idade.</li>
	<li>Aplique/pulverize 70% de etanol no abdômen e exponha a cavidade abdominal inferior usando tesoura limpa e fórceps.</li>
	<li>Localizar almofada de gordura epidídica (proximal/ligada a testículos). Segure a junção entre testículos e almofada de gordura epidídica com fórceps contundentes e puxe suavemente para liberar a almofada de gordura epidídica.</li>
	<li>Remova os testículos cortando usando uma tesoura e incubar a almofada de gordura epidídica em 5 mL de HBSS suplementada com 3% de BSA em um tubo cônico de 50 mL em temperatura ambiente por 15 minutos.</li>
	<li>Centrifugar a 150 x g por 7 min a 4 °C.</li>
	<li>Pegue a almofada de gordura epidímica flutuante do tubo cônico de 50 mL e pique finamente o tecido usando uma tesoura limpa.</li>
	<li>Adicione 200 μL de solução de colagenase 2% em 3,8 mL de HBSS em um tubo de cultura de 13 mL. Adicione o tecido picado e incubar em uma incubadora rotativa a 5 rpm por 1h a 37 °C. Em vez de uma incubadora rotativa, a incubadora de cultura celular a 37 °C pode ser usada alternativamente com agitações ocasionais.</li>
	<li>Transfira o conteúdo inteiro para um tubo cônico de 50 mL e adicione 10 mL de meio de neutralização. Misture suavemente invertendo o tubo 2-3 vezes.</li>
	<li>Prepare outro tubo cônico de 50 mL equipado com um coador de células de 70 μm. Filtre o tecido digerido no novo tubo usando o coador celular.</li>
	<li>Transfira o fluxo para um tubo cônico de 15 mL e centrífuga a 350 x g por 10 min a 4 °C.</li>
	<li>Remova cuidadosamente as pelotas de células supernascidas e resuspendas com 5 mL de DPBS. Centrifugar a 350 x g por 10 min a 4 °C.</li>
	<li>Remova cuidadosamente o supernascimento e adicione 50 μL de tampão de citometria de fluxo. Misture bem por pipetar suavemente e mantenha as células no gelo. Devido ao volume das pelotas celulares e à pequena quantidade de supernanato residual, o volume total de células no tampão de citometria de fluxo será superior a 50 μL (aproximadamente 100 μL).</li>
</ol>3. Rotulagem de anticorpos e fluorescência ativada de classificação celular (FACS)
<ol><li>Depois de misturar bem as células com tubulação suave, transfira 54 μL da suspensão celular para um tubo de microcentrifuge de 1,7 mL. Adicione 6 μL de reagente de bloqueio fcr e misture bem por pipetar suavemente. Incubar a 4 °C por 10 min. Salve as células restantes depois de tomar 54 μL e mantenha-as no gelo para usar como um controle não manchado na etapa 3.5 e passo 3.6.</li>
	<li>Durante a incubação na etapa 3.1, prepare um coquetel de anticorpos combinando anticorpos de 11 μL cada um de anticorpos anti-Sca1-APC, anti-Ter119-FITC, anti-CD31-FITC e anticorpos anti-CD45-FITC em um tubo de microcentrifuuge. Misture bem com tubos suaves e mantenha no gelo protegido da luz.</li>
	<li>Após a incubação com o reagente de bloqueio fcr por 10 minutos, adicione 40 μL do coquetel de anticorpos e misture bem por pipetação suave. Incubar a 4 °C por 10 min.</li>
	<li>Adicione 500 μL de tampão de citometria de fluxo suplementado com 1 μg/mL de DAPI nas células manchadas. Misture bem por células de tubulação suaves e filtros usando um tubo de ensaio de 5 mL com tampa de encaixe do filtro celular. Mantenha células no gelo.</li>
	<li>Adicione 500 μL de tampão de citometria de fluxo às células remanescentes na etapa 3.1 e misture bem por tubulação suave. Células filtradoras usando um tubo de ensaio de 5 mL com tampa de encaixe do filtro celular. Mantenha essas células no gelo e use como um controle não manchado na etapa seguinte.</li>
	<li>Leve as células manchadas e o controle não manchado para análise ou instrumento de classificação FACS.<ol>
<li>Identifique e isole a população APC+/FITC-/DAPI- com estratégias de gating mostradas na Figura 1. Essas células representam células viável sca1+/Ter119-/CD31-/CD45. Detritos e agregados de células são esgotados usando parcelas para a frente (FSC) e dispersão lateral (SSC). Em seguida, o DAPI- população é fechada, seguido de gating de APC+/FITC- população. Os controles não manchados devem ser usados para auxiliar na definição de parâmetros de gating.</li>
		<li>Colete as células em 500 μL de tampão de citometria de fluxo em um tubo de microcentrifuge de 1,5 mL. Seguindo em frente, todos os procedimentos devem ser realizados em condições estéreis para minimizar uma contaminação. Aproximadamente 50.000 – 100.000 células são coletadas de um rato.</li>
	</ol>
</li>
	<li>Conte células usando um hemócito para avaliar a viabilidade celular. Uma vez que os agregados celulares podem interferir com novas análises a jusante, a presença de agregados celulares também é avaliada para garantir uma presença mínima (<5% do número celular viável) desses agregados.</li>
</ol>

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O sequenciamento de RNA de célula única (scRNA-seq) está rapidamente ganhando tração como uma ferramenta poderosa para estudar simultaneamente diversas populações celulares no nível único celular. Devido aos altos custos associados à preparação da amostra e ao sequenciamento de alto rendimento, é imperativo otimizar as entradas celulares (alta viabilidade e pureza) para aumentar a probabilidade de sucesso experimental. Alguns protocolos de preparação celular dependem da remoção de células mortas e detri...

O tecido adiposo desempenha um papel fundamental no metabolismo energético. O excesso de energia é armazenado na forma de lipídios, e o tecido adiposo é capaz de expansão ou retração significativa, dependendo do estado nutricional e da demanda energética. A expansão do tecido adiposo pode resultar de um aumento no tamanho do adipócito (hipertrofia) e/ou de um aumento no número de adipócitos (hiperplasia); este último processo fortemente regulado pela proliferação e diferenciação das células progenitorasadiposas 1,2. Durante a obesidade, o tecido adiposo se expande excessivamente, e a disfunção tecidual – incluindo hipóxia, inflamação e resistência à insulina – muitas vezes desenvolve3,4. Essas complicações são fatores de risco para muitas doenças crônicas, incluindo hipertensão arterial, diabetes, doenças cardiovasculares, acidente vascular cerebral e câncer5. Assim, limitar a expansão descontrolada do tecido adiposo e mitigar as patologias do tecido adiposo são prioridades de pesquisa biomédica. Durante a expansão do tecido adiposo, as células-tronco derivadas do tecido adiposo residente (ASCs) proliferam e diferenciam-se sequencialmente em pré-adipócitos (células progenitoras comprometidas) e, em seguida, em adipócitos maduros6. Estudos recentes de sequenciamento de RNA unicelular (scRNA-seq) mostram que essas populações de células progenitoras adiposas (APC) (ASCs e preadipócitos) apresentam heterogeneidade molecular e funcional substancial7,8,9,10,11,12. Por exemplo, as ASCs apresentam uma capacidade de diferenciação adipogênica reduzida, ao mesmo tempo em que apresentam maiores capacidades de proliferação e expansão, em comparação com os pré-apócitos7. Outras diferenças moleculares são relatadas nas populações asc e pré-doença, embora a relevância funcional dessas diferenças ainda não esteja clara7. Juntos, esses dados destacam a complexidade do pool de células progenitoras adiposas e ressaltam a necessidade de desenvolver e padronizar ferramentas para melhor entender e manipular essas populações celulares críticas.
Este protocolo detalha o isolamento de populações de células progenitoras de alta viabilidade Sca1+ adiposas de blocos de gordura epidídica de camundongos que são adequados para análises sensíveis a jusante, incluindo estudos unicelulares (scRNA-sequenciamento) e cultura celular. O isolamento e dissociação de almofadas de gordura epidídica foi realizado como descrito anteriormente7,13 com pequenas modificações que melhoram a viabilidade de APCs isolados. Em suma, células dissociadas de almofadas de gordura epidídimo são manchadas com anticorpos contra Sca1, um marcador para ambos os ASCs e preadipócitos6,7, e outros marcadores de linhagem (Lin): Ter119 (células eritróides), CD31 (células endoteliais) e CD45 (leucócitos). As células Viável Sca1+/Ter119-/CD31-/CD45-/DAPI- são então classificadas por fluorescência ativada de classificação celular (FACS). É importante ressaltar que este protocolo foi validado pelo isolamento e análise bem-sucedidos de células progenitoras viáveis Sca1+/Lin- adiposas relatadas em um estudo recente de sequenciamento de RNA de célula única que identificou subpopulações funcionalmente heterogêneas dentro de ASCs e preadipócitos7.

<table><tbody><tr><td>1.7 mL microcentrifuge tube</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>13 mL culture tube</td><td>Thermo Fisher Scientific</td><td>50-809-216</td><td></td></tr><tr><td>15 mL conical tube</td><td>Greiner Bio-one</td><td>188 271</td><td></td></tr><tr><td>5 mL test tube with cell strainer snap cap</td><td>Thermo Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner Bio-one</td><td>227 261</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Thermo Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Anti-CD31-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-519</td><td></td></tr><tr><td>Anti-CD45-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-491</td><td></td></tr><tr><td>Anti-Sca1-APC antibody</td><td>Miltenyi Biotec</td><td>130-102-833</td><td></td></tr><tr><td>Anti-Ter119-FITC antibody</td><td>Miltenyi Biotec</td><td>130-112-908</td><td></td></tr><tr><td>BSA</td><td>Gold Biotechnology</td><td>A-420-500</td><td></td></tr><tr><td>Collagenase type II</td><td>Thermo Fisher Scientific</td><td>17101-015</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>F-12 medium</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td></td></tr><tr><td>FcR blocking reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Miltenyi Biotec</td><td>130-093-233</td><td></td></tr></tbody></table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

Obesidade e distúrbios metabólicos, como diabetes, doenças cardíacas e câncer, estão todos associados à dramática remodelação do tecido adiposo. As células progenitoras adiposas residentes em tecido (APCs) desempenham um papel fundamental na homeostase tecidual e podem contribuir para a patologia do tecido. O uso crescente de tecnologias de análise celular única – incluindo sequenciamento de RNA unicelular e proteômica unicelular – está transformando o campo de células-tronco/progenitora, permitindo uma resolução sem precedentes de mudanças individuais de expressão celular no contexto de mudanças populacionais ou teciduais. Neste artigo, fornecemos protocolos detalhados para dissecar tecido adiposo epidímico do camundongo, isolar células derivadas de tecido adiposo único e realizar a triagem celular ativada de fluorescência (FACS) para enriquecer para Sca1+/CD31 viável-/CD45-/Ter119- APCs. Esses protocolos permitirão que os pesquisadores preparem APCs de alta qualidade adequados para análises a jusante, como sequenciamento de RNA de célula única.

Camundongos FVB machos de quatro meses de idade foram usados neste experimento. Após a exclusão de detritos e dobras utilizando lotes FSC/SSC, células viáveis (DAPI- população) foram fechadas, seguidas pela seleção de APC+/FITC- população (Figura 1). Os portões DAPI, APC e FITC foram desenhados com base no controle não manchado. As estratégias de gating são mostradas na Figura 1.
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Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Apresentamos um método simples para isolar células progenitoras adiposas altamente viáveis de almofadas de gordura epidímica do rato usando a triagem celular ativada por fluorescência.

Preparação de células progenitoras adiposas adiposas de tecidos adiposos epidímicos do rato

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

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5.6K visualizações.  Mayo Clinic. Apresentamos um método simples para isolar células progenitoras adiposas altamente viáveis de almofadas de gordura epidímica do rato usando a triagem celular ativada por fluorescência.

Vídeo: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Geração de adiposo humano Stem Cells através Dedifferentiation de adipócitos maduros em Culturas de teto

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Biologia do Desenvolvimento

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Isolamento e diferenciação de células tronco derivadas de tecido adiposo de Suínos Subcutânea tecidos adiposos

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

<meta charset="utf8"></head><body>Isolamento e processamento de adipócitos brancos murinos para análises de transcriptoma e epigenoma

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Isolamento e processamento de adipócitos brancos murinos para análises de transcriptoma e epigenoma

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genética

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

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

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Imunologia e Infecção

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

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

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.

Isolamento de Subpopulações de Células Estromais Adipogênicas e Fibro-Inflamatórias de Depósitos Adiposos Intra-Abdominais Murinos

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Isolamento e diferenciação de preadipócitos brancos e marrons primários de camundongos recém-nascidos

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Biologia

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

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

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.

Adipócitos diferenciados de camundongos em cultura primária: um modelo de resistência à insulina

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Cultura de pré-adipócitos isolados de tecido adiposo perivascular murino

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Autor em destaque: A importância do isolamento, cultura e indução adipogênica de pré-adipócitos derivados de SVF do tecido adiposo perivascular de camundongos 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Preparação de células progenitoras adiposas adiposas de tecidos adiposos epidímicos do rato

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