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>

<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 border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="799">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">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 ...

preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Research

JoVE Journal

Biology

5.5K 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

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolamento manual de células-tronco adiposo-derivadas de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Biologia

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Capacidade de diferenciação de células progenitoras de aorta humana Perivascular adiposo

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Identificação e dissecção de diversos depósitos de Adipose de camundongo

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Isolamento e Identificação de Células Endoteliais Vasculares de Depósitos Adiposos Distintos para Aplicações a Jusante

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Isolamento e caracterização de células-tronco derivadas do tecido adiposo

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Autor em destaque: Isolamento e identificação de células-tronco mesenquimais derivadas do tecido adiposo de ratos Sprague Dawley  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Isolamento e Identificação de Células-Tronco Mesenquimais Derivadas do Tecido Adiposo de Ratos Sprague Dawley

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

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.