Este trabalho foi financiado pelo National Institutes of Health Grant 5KO8DK085136-02 a JY.

1. Obter Colon Tissue Mouse Um protocolo para realizar experimentos com animais, detalhando os fundamentos e objetivos da pesquisa, foi submetido e aprovado pelo nosso Departamento de Pesquisa institucional Supervisão Animal. <ol><li> Eutanásia o mouse com isoflurano inalado seguido por deslocamento cervical. </li><li> Faça uma incisão na linha média ventral, retraia o intestino delgado de distância e identificar o cólon. Recolha a bexiga e o útero (se presente) lateralmente e identificar o osso púbico e cortar com uma tesoura fina para expor o recto. </li><li> Dissecar o mesentério dorsal do recto fora do cólon e mobilizar o cólon a partir do ânus ao ceco, e cortar através de que a desconectar o cólon a partir do intestino delgado. </li><li> Coloque as amostras em solução salina tamponada com fosfato gelada (PBS). </li><li> Encha uma seringa de 10 ml com PBS gelado e lavar o cólon lúmen várias vezes para limpar o seu conteúdo, em seguida, corte o ope cólonn longitudinalmente com uma tesoura. </li><li> Coloque o cólon num tubo cónico de 50 ml contendo 20 ml de PBS gelado. O cólon não tem de ser colocado em qualquer orientação particular. Lavar o cólon, substituindo o PBS até que o líquido é claro, sem material particulado. </li></ol> Humano Um protocolo para obtenção de tecido humano de pacientes cirúrgicos, detalhando a importância de se obter este tecido, bem como uma avaliação dos riscos e benefícios potenciais, foi submetido e aprovado pelo nosso escritório institucional do Programa de Proteção Human Research. <ol><li> Colaboração na investigação com um cirurgião colorretal é necessário para ser capaz de obter o tecido humano para a sua pesquisa. Um protocolo para obtenção de tecido de cólon humano deve ser submetido e aprovado pelo seu conselho de revisão institucional. </li><li> O consentimento informado é obtido a partir do paciente antes do procedimento cirúrgico. </li><li> O cirurgião irá realizar uma ressecção do cólon no standmoda ard. Uma vez que o cólon foi removido a partir do paciente, o espécimen é imediatamente feita a patologia. A 0,5 em secção de espessura total da parede do cólon não são necessários para a avaliação patológica é fornecida e é imediatamente colocado em PBS gelado. Este espécime não deve incluir o mesentério do cólon, e apêndices epiplóico devem ser removidos, bem como, uma vez que a presença de gordura vai afetar o processo de digestão. </li></ol> 2. Desnudar células epiteliais <ol><li> Incubar todo o cólon do rato em 25 ml de EDTA 5 mM em HBSS (temperatura ambiente) em um tubo de 50 ml. Colocar o tubo cónico de 37 ° C agitando banho de ar (250 rpm) durante 15 min. Após 15 minutos, cuidadosamente deitar fora o líquido e encher o tubo com 25 ml de EDTA 5 mM, para repetir um total de cinco lavagens. Para cólon humano, após a obtenção de uma faixa de parede do cólon ½ polegada a partir do patologista cortar o cólon com uma tesoura em quatro partes de igual tamanho. Coloque duas das peças em um tubo de 50 ml, ecolocar as restantes duas peças de tecido do cólon para um tubo cónico de 50 ml separado. Em seguida incubar com 25 ml de EDTA 5 mM em HBSS e executar lavagens, tal como descrito acima. </li><li> Lavar o cólon duas vezes em 20 ml de PBS gelado. </li><li> Colocar o tecido do cólon do rato em um novo tubo de 50 ml contendo 20 ml de meio RPMI-5, 10 L de dispase, e 2,000 U de colagenase D (temperatura ambiente). Para o tecido do cólon humano, usar o dobro da quantidade de dispase e colagenase (20 U dispase, 4000 U de colagenase D). </li><li> Colocar o tubo num banho de ar de 37 ° C agitando orientada verticalmente (Se for colocado horizontalmente, há muito arejamento e danos celulares). Agitar a 250 rpm, durante até 60 min. Após a exposição ao dispase e colagenase D, o tecido do cólon vai começar a digerir o tecido do cólon e começará a procurar fibroso. Isso geralmente leva cerca de 30 min. Quando o cólon tem esta aparência, remover o cólon das enzimas. </li><li> Agitar cada tubo de cima e para baixo 2-4 vezes para romper o tecido. PEllet o tecido a 200 xg numa centrifugadora de mesa (4 ° C) durante 5 min. Despeje cuidadosamente o sobrenadante e descartar. </li></ol> 3. Isolamento e Cultura de miofibroblastos <ol><li> Ressuspender a cada sedimento por adição de 10 ml de tampão de lise ACK (47 ° C). Centrifugar novamente a 200 x g durante 5 minutos (4 ° C), deitar fora e desprezar o sobrenadante. </li><li> Ressuspender a cada sedimento por adição de 10 ml de RPMI-5 com uma pipeta. </li><li> Passa metade da suspensão de células (5 ml) através de uma peneira de malha 70 mM com uma pipeta de 10 ml para uma placa de 100 mm tratadas com TC. Em seguida, adicionar um adicional de 15 ml de RPMI-5. Repetir com os restantes 5 ml de suspensão de células para um prato de 100 mm diferente tratados com TC. Um rato de cólon, portanto, deu dois pratos tratados com TC 100 mm. Tira de tecido de cólon humano Uma ½ polegada irá produzir um total de quatro placas tratadas com TC de 100 mm. </li><li> Colocar as placas de cultura de tecido em 10% de CO2 a 37 ° C. As condições de cultura são as mesmas fou mouse e células primárias humanas. </li><li> Após 3 h, lavar suavemente as células não aderentes, com duas alterações de HBSS. O flutuante de detritos devem ser lavadas delicadamente fora. As células aderentes são compostas por células epiteliais, macrófagos e miofibroblastos. Uma semana após a semeadura, apenas miofibroblastos dividir, macrófagos e células epiteliais senesce após a primeira passagem. Adicionar fresco RPMI-5 e cultivar as células em cultura de células. </li><li> As células são passadas por tripsinização, durante 5 min e são divididas 2:1. </li></ol> Abreviaturas HBSS: solução de sal equilibrada de Hank; RPMI-5: meios de Roswell Park Memorial Institute; ACK: Cloreto de amónio--potássio; FCS: soro fetal de vitelo; TC-tratados: cultura de tecidos tratados Soluções Tampão de lise ACK - (4,15 g de NH4Cl, 0,5 g de KHCO3, 18,6 mg de Na 2 EDTA, 400 ml de H 2 O, ajustar o pH a 7,2-7,4 com HCl 1 N). Filtro esterilizadore através de um filtro de 0,2 um e armazenar a 4 ° C. RPMI-5 - (a perfazer 500 ml: 454,5 ml de RPMI, FCS 25 ml, 5 ml de 200 mM de L-glutamina, 5 mL de HEPES 1 M, pH 7,4, 5 ml 100 mM de piruvato de sódio, 5 ml de 100x Pen-Strep, 500 ml de 50 mM B-ME em PBS).

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Autores têm nada a revelar.

A técnica geral é semelhante ao usar o mouse ou o cólon humano. Esta técnica também pode ser usada para isolar os miofibroblastos do intestino delgado. Detalhes específicos para o isolamento de miofibroblastos de 1. rato e 2. cólon humano são discutidos abaixo. 1. Rato Colon Irrigação do cólon do rato é facilitada pela fixação de um 4 em 16 L Popper agulha de extremidades cegas para uma das extremidades do cólon. Isso também ajuda quando o corte do c...

A regulação da função do trato gastrointestinal (GI), envolve interações dinâmicas complexas entre a camada mucosa e seu mesênquima subjacente. Estas interacções normalmente servem para manter a homeostase, mas também pode levar ao desenvolvimento de estados de doença, em condições patológicas 1. Os miofibroblastos são uma subpopulação de células estromais localizados subjacente à camada epitelial que comunicar de uma forma parácrina com áreas subpopulações de células para regular um número de processos críticos do tracto GI que incluem regeneração das mucosas, reparação, e fibrose 2. A linha celular de 18Co é um exemplo de uma linha celular humana do cólon miofibroblastos que tem sido amplamente utilizado para estudar a função de miofibroblastos porque partilha muitas das características de primário em miofibroblastos situ. Estes incluem a expressão de proteínas de um-actina de músculo liso (α-SMA) e vimentina, bem como a expressão de uma sériede receptores de superfície celular, tais como o receptor do factor de crescimento epidérmico, ou receptores de 3,4 ácido lisofosfatídico. Devido a estas características ultra-estruturais comuns, bem como similaridades na função biológica 5, a linha de células de 18Co tem sido amplamente utilizado para estudar a função de miofibroblastos no contexto de muitos estados de doença, tais como a doença inflamatória do intestino ou 3,6 cancro colorectal. No entanto, existem limitações inerentes e preocupações na utilização de uma linha celular. Estes incluem a instabilidade genotípica ao longo do tempo que diferencia as linhas celulares a partir de células primárias, alterando o fenótipo e função biológica da célula, incluindo as taxas de crescimento e as interacções com outras populações de células. As linhas celulares também não possuem os componentes normais do microambiente GI (epitelial, estromal, e componentes vasculares), que é uma limitação importante à sua utilização. Portanto, as conclusões da pesquisa linhagem celular experimental requer uma validação adicional para reduzir o risk de má interpretação. Miofibroblastos primárias podem ser obtidas a partir de tecido de cólon humano e de ratinho para confirmar os resultados experimentais identificados em uma linha de células 7. O método foi descrito originalmente por Mahida et al. 8, e o nosso protocolo é uma das várias técnicas bem definidas que podem ser utilizadas para isolar os miofibroblastos primários de ratinho e de cólon humano 9. Para qualquer modelo do rato da doença colônica, esta técnica pode ser usada para isolar myofibroblasts principais para estudar a forma como eles interagem com as células vizinhas ou contribuir para tanto normal ou patológica GI processa 10,11. Esta técnica pode ser usada para estudar como miofibroblastos contribuir para a cicatrização da mucosa, fibrose, e o desenvolvimento de adenomas e carcinomas. Miofibroblastos primárias também pode ser obtido a partir de tecido de cólon humano que foi ressecado, no momento da operação de benigna (doença inflamatória do intestino, estenoses, diverticulite) ou maligno cCONDIÇÕES. As células primárias isoladas a partir de tecido de cólon humano e de ratinho podem ser cultivadas em cultura celular e utilizada ao longo de um número limitado de passagens.

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

O miofibroblastos é uma célula estromal do tracto gastrointestinal (GI) que tem vindo a ganhar uma atenção considerável para o seu papel fundamental em muitas funções gastrointestinais. Enquanto várias linhas de células de miofibroblastos são comercialmente disponíveis para estudar estas células in vitro, os resultados da pesquisa de uma linha de células expostas a condições de cultura de células experimentais têm limitações inerentes devido à natureza excessivamente redutora do trabalho. Utilização de miofibroblastos primárias oferece uma grande vantagem em termos de resultados experimentais, confirmando identificados numa linha celular. Isolamento de miofibroblastos primárias a partir de um modelo animal, permite o estudo dos miofibroblastos, em condições que mais de perto simulam o estado da doença a ser estudada. Isolamento de miofibroblastos primários de tecido do cólon humano oferece, sem dúvida, os dados experimentais mais relevantes, uma vez que as células vêm diretamente de pacientes com a doença de base. Nós descrevemos uma técnica bem estabelecida, que pode ser utilized isolar myofibroblasts primários do mouse e do tecido do cólon humano. Estas células isoladas foram caracterizadas como actina alfa de músculo liso e vimentina-positivas, e desmina-negativos, de acordo com miofibroblastos subepiteliais intestinais. Células de miofibroblastos primárias podem ser cultivadas em cultura de células e utilizadas para fins experimentais mais de um número limitado de passagens.

Uma vez isolado, miofibroblastos humanas primárias podem ser cultivadas em cultura celular e utilizada ao longo de um número limitado de passagens (até à passagem 4). Estas células são caracterizados como sendo um liso-actina de músculo e vimentina positivo, e desmina negativa (Figura 1A), consistente com os miofibroblastos subepiteliais intestinais 5,7. Eles também têm uma estreladas morfologia característica (Figura 1B). Miofibroblastos primários podem então ser...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

O miofibroblastos é uma célula estromal influente do tracto gastrointestinal, que regula os processos fisiológicos importantes em estados normais e de doença. Descrevemos uma técnica que permite o isolamento de miofibroblastos primários de rato e tanto o tecido do cólon humano, o que pode ser utilizado para experimentação in vitro.

Isolamento de primárias Miofibroblastos de Mouse e tecido do cólon humano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

isolation-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

Research

JoVE Journal

Biology

19.5K visualizações.  UCLA.  O miofibroblastos é uma célula estromal influente do tracto gastrointestinal, que regula os processos fisiológicos importantes em estados normais e de doença. Descrevemos uma técnica que permite o isolamento de miofibroblastos primários de rato e tanto o tecido do cólon humano, o que pode ser utilizado para experimentação in vitro. 

Vídeo: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Isolamento Núcleos neuronais de Tecidos Humanos Postmortem Cérebro

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Biologia

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolamento de células imunes de tumores primários

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e Análise Funcional de mitocôndrias de células cultivadas e mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Isolamento de Miofibroblastos de Mouse e esôfago humano

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolamento e Caracterização de Fibroblastos Cardíacos Adultos e Miofibroblastos

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

Geração de Monócamas Epiteliais de Cólon Primário Murine de Criptas Intestinais

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...