Este trabalho foi parcialmente apoiado por um Fundo-A-Fellow subvenção da Myotonic Dystrophy Foundation (MDF-FF-2014-0013) eo Cientista Development Grant da American Heart Association (16SDG29320001).

Os ratinhos transgénicos de tipo selvagem e Nestin-GFP foram alojados na instalação animal na Universidade de Minnesota. Todos os procedimentos experimentais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade de Minnesota e estavam de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório. 1. Dissecção muscular e isolamento de célula única <ol><li> Euthanize camundongos adultos (6-10 semanas, ambos do sexo masculino e feminino) com tribromoetanol (250 mg / kg, ip) e esterilizar a pele do abdômen com etanol 70%. NOTA: O tribromoetanol foi usado aqui em vez de cetamina para anestesia / eutanásia, já que a ketamina é conhecida por interagir com os receptores NMDA, o que poderia potencialmente ter um impacto no estudo. </li><li> Coloque os ratos na posição supina e use um bisturi para fazer uma incisão horizontal na pele abdominal. Descasque a pele com a mão, puxando em direções opostas para expor os músculos dos membros posteriores. </li><li> ColecionarT os músculos de ambos os membros posteriores usando fórceps e tesoura. Armazená-los em solução salina tamponada com fosfato (PBS) estéril suplementada com 1% de penicilina-estreptomicina (P / S) em gelo. </li><li> Lavar os músculos do membro posterior dissecados em PBS gelado suplementado com P / S a 1% duas vezes e transferi-los para uma placa estéril de 10 cm. </li><li> Cuidadosamente dissecar os nervos, vasos sanguíneos e tecido conjuntivo dos músculos usando pinças e tesoura sob um microscópio de dissecção em 2X ampliação. </li><li> Cortar finamente e picar os músculos em pedaços pequenos (1-2 mm 3 ) usando tesouras estéreis e lâminas. Se necessário, adicionar uma pequena quantidade de Dulbecco Modified Eagle Medium (DMEM) para garantir que os músculos não são secos. </li><li> Quebrar mecanicamente o tecido por pipetagem para cima e para baixo através de uma pipeta serológica de 10 ml 10 vezes. </li><li> Adicionar a solução de digestão recentemente preparada (DMEM suplementado com colagenase de tipo 2 a 0,2%) à mistura. Incubar a 37 ° C por 2 h com gentlAgitação a 35 revoluções / min. </li><li> Triturar utilizando uma agulha de 18 G para homogeneizar a mistura. Em seguida, centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e depois ressuspender o sedimento em 0,25% de tripsina / EDTA. Incubar a 37 ° C durante 10 min. Repita o passo de centrifugação mais duas vezes. </li><li> Adicionar 10 mL de DMEM suplementado com 20% de soro fetal bovino (FBS) à solução e centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em tampão de lise de células vermelhas do sangue (NH4Cl 155 mM, KHCO3 10 mM e EDTA 0,1 mM). </li><li> Centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em tampão de separação (HEPES 20 mM, pH 7,0, EDTA 1 mM e BSA a 1% em PBS livre de Ca / Mg2 + , pH 7,0). Filtrar a mistura através de um filtro de células de 40 um para obter uma suspensão de célula única. </li><li> Centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em 1 mL de tampão de separação. </li><li> Conte oNúmero de células com um hemocitômetro e diluir a suspensão de célula única para 5 x 10 6 / mL em tampão de triagem. </li></ol> 2. Coloração e Classificação de Células <ol><li> Preparar os controlos ea amostra conforme descrito na Tabela 1 . Mancha a suspensão de célula única com os respectivos anticorpos em gelo durante 30 min, como descrito na Tabela 1 . </li><li> Centrifugar a 500 xg durante 5 min e lavar os pellets duas vezes com tampão de separação. </li><li> Adicionar DAPI às soluções de célula única, como indicado na Tabela 1. Utilizar 5 μg / mL de DAPI (concentração final) para o controlo de cor única DAPI e 1 μg / mL de DAPI para o controlo de PDGFRβ-PE-FMO e para a amostra. Manter todos os tubos em gelo durante todo o experimento. </li><li> Ative o classificador e o software. Digitalize e insira um chip de classificação de 100 μm quando solicitado. </li><li> Execute a configuração automática ( ie, alinhamento de chip , calibração de gotículas, calibração de fluxo lateral e calibração de atraso de classificaçãoOn) carregando os grânulos de configuração automática quando solicitado. </li><li> Quando a configuração automática estiver concluída, vá para a guia &quot;Experiência&quot;, clique em &quot;Novo&quot; e selecione o &quot;Modelo em Branco&quot; em &quot;Modelos Públicos&quot;. </li><li> Em &quot;Configurações de medição&quot;, digite &quot;DAPI&quot; para &quot;FL1&quot;, &quot;Nestin-GFP&quot; para &quot;FL2&quot; e &quot;PDGFRβ&quot; para &quot;FL3.&quot; Desmarque as caixas de &quot;FL4&quot; - &quot;FL6&quot;. </li><li> Marque as caixas para ativar os lasers 405, 488 e 561 e clique em &quot;Criar Experiência Nova&quot;. </li><li> Selecione a opção &quot;Start Compensation Wizard&quot; e siga as instruções do software &quot;Compensation Wizard&quot; para configurar a compensação. <ol><li> Carregar o controle sem cor e clique em &quot;Iniciar&quot;. Clique em &quot;Detector &amp; Threshold Settings&quot; e ajuste o ganho do sensor dos detectores FSC e BSC para colocar a população na escala. </li><li> de AnúnciosApenas os níveis de ganho dos canais de fluorescência FL1-FL3 para colocar as populações negativas no lado esquerdo dos histogramas. Clique no botão &quot;Gravar&quot; para gravar os dados. </li><li> Carregar controles de uma cor um por um quando solicitado. Clique em &quot;Iniciar e Gravar&quot; para gravar os dados. Ajustar os portões para as populações positivas nos histogramas. Clique em &quot;Avançar&quot;. </li><li> Vá para &quot;Calcular matriz&quot; na guia &quot;Compensação&quot; e clique em &quot;Calcular&quot; no painel &quot;Calcular configurações de compensação&quot; para executar a compensação. Clique em &quot;Concluir&quot; para sair do &quot;Assistente de compensação&quot;. </li></ol></li><li> Carregue o controle PDGFRβ-PE-FMO e clique em &quot;Iniciar&quot;. Desenhe um portão de polígono (Portão A) em torno das células de interesse sob o gráfico &quot;Todos os Eventos&quot;. </li><li> Clique duas vezes dentro do portão A para criar um gráfico filho. Altere o eixo Y para DAPI e desenhe uma porta poligonal (Gate B) em torno de células vivas (DAPI low ). Clique duas vezes em iNside Gate B para criar um enredo filho. Mude o eixo X para FSC-H e o eixo Y para FSC-W e desenhe uma porta poligonal (Gate C) em torno dos singlets para eliminar dupletos. </li><li> Clique duas vezes dentro do Gate C para criar um gráfico filho. Mudar o eixo X para Nestin-GFP e o eixo Y para PDGFRβ-PE. Clique no botão &quot;Gravar&quot; para gravar os dados. </li><li> Carregue a amostra e repita os passos 2.11-2.12. Após a gravação, clique em &quot;Pausar&quot; para preservar a amostra. </li><li> Definir os limites de passagem para as células PDGFRβ + e Nestin-GFP + com base no controlo PDGFRβ-PE-FMO. Draw portões para o PDGFRβ + Nestin-GFP - e PDGFRβ + Nestin-GFP + populações. </li><li> Em &quot;Método de classificação&quot;, selecione &quot;Tubos de 2 vias&quot; e atribua as células &quot;PDGFRβ + Nestin-GFP - &quot; e &quot;PDGFRβ + Nestin-GFP + &quot; aos tubos coletores esquerdo e direitoEspecificamente. Montar os tubos de recolha de 15 mL de tampão de triagem na fase de recolha e clicar no botão &quot;Carregar colecção&quot;. </li><li> Clique no botão &quot;Continuar&quot; para manter a amostra em execução. Clique em &quot;Start Sort&quot; para coletar as células PDGFRβ + Nestin-GFP - (tipo I) e células PDGFRβ + Nestin-GFP + (tipo II pericytes). </li></ol> 3. Análises pós-triagem <ol><li> Centrifugar as células classificadas a 500 xg durante 5 min, ressuspender a pastilha em 1 mL de meio pericito (ver Tabela de Materiais ) e contar a densidade celular utilizando um hemocitómetro. </li><li> Os pericitos tipo I e tipo II de sementes em lamínulas revestidas com poli-D-lisina (PDL) a ~ 1 x 10 4 células / cm2. Crescer em meio pericíto durante 3 dias a 37 ° C com 5% de CO2. </li><li> No dia 3, examinar a morfologia do pericito (sob contraste de fase) e endógena Nestin-GFP expressão utilizando um micro fluorescenteÂmbito (laser de excitação: 488 nm, filtro de excitação: 470/40 nm, e filtro de emissão: 515/30 nm). Tire as imagens com um objetivo de 20X (0,45 NA). </li><li> Substituir o meio pericítico por meio adipogênico (meio basal MSC + suplemento estimulante adipogênico) e miogênico (DMEM + 2% soro de cavalo) para iniciar a diferenciação adipogênica e miogênica, respectivamente, conforme descrito anteriormente 20 . Alterar o meio a cada 2-3 dias. </li><li> Fixar as células no dia 17 (14 dias após a diferenciação adipogénica / miogénica) em paraformaldeído a 4% (PFA) durante 20 min à temperatura ambiente. NOTA: Cuidado, PFA é um carcinógeno. </li><li> Realizar imunocitoquímica contra perilipina (marcador de adipócito) e S-miosina (marcador de miotubo maduro / miofibra), conforme descrito em publicações anteriores 19 , 20 . <ol><li> Lavar as células fixas 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Adicionar tampão de bloqueio (PBSSuplementado com soro de burro a 5%, BSA a 3% e Triton X-100 a 0,3%) e incubar à temperatura ambiente durante 1 h. </li><li> Incubar as células com anticorpos anti-perilipina (2 μg / mL) e / ou anti-S-miosina (2 μg / mL) a 4 ° C durante a noite. </li><li> Lavar as células 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Incubar as células com anticorpos anti-coelho anti-coelho (4 μg / mL) e / ou Alexa555 anti-rato (4 μg / mL) de burro Alexa 555 à temperatura ambiente durante 1 h. </li><li> Lavar as células 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Monte as células imunocoradas com meio de montagem contendo DAPI (ver tabela de materiais). Examine a expressão de perilipina e S-miosina utilizando um microscópio fluorescente (laser de excitação: 543 nm, excitação de 540/45 nm e emissão de 600/50 nm) e tome imagens sob um objetivo 40X (0,60 NA). </li></ol></li></ol>

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Os pericitos são células perivasculares multipotentes 22 , 23 localizadas na superfície abluminal dos capilares 21 , 26 . Nos músculos esqueléticos, os pericitos são capazes de diferenciar ao longo das vias adipogénicas e / ou miogénicas 19 , 20 , 24 . Estudos recentes revelaram duas subpopulações de p...

A distrofia muscular é uma desordem músculo-degenerativa que não tem nenhum tratamento eficaz até agora. O desenvolvimento de terapias que promovem a regeneração de tecidos tem sido sempre de grande interesse. A regeneração e reparação dos tecidos após os danos dependem das células estaminais / células progenitoras residentes 1 . As células satélites são células precursoras miogénicas comprometidas que contribuem para a regeneração muscular 2 , 3 , 4 , 5 , 6 , 7 . O seu uso clínico, no entanto, é dificultado pela sua migração limitada e baixa taxa de sobrevivência após a injecção, bem como pela sua diminuição da capacidade de diferenciação após a amplificação in vitro 8 , 9 , 10 , 11 . Além do satelliTe células, os músculos esqueléticos também contêm muitas outras populações de células com potencial miogénico 12 , 13 , 14 , 15 , 16 , tais como as células intersticiais positivas ao receptor de factor de crescimento derivado de plaquetas beta (PDGFRp). Há evidências que demonstram que as células PDGFRβ + derivadas de músculo são capazes de se diferenciar em células miogênicas e melhorar a patologia e a função muscular 14 , 17 , 18 , 19 , 20 . O PDGFRβ rotula predominantemente os pericitos 21 , que são células perivasculares com pluripotência 22 , 23 . Além de PDGFRβ, muitos outros marcadores, incluindo Neuron-Glial 2 (NG2) e CD146, também são usados ​​para iDentify pericytes 21 . Deve-se notar, no entanto, que nenhum desses marcadores é pericítico-específico [ 21] . Estudos recentes revelaram dois subtipos de pericitos musculares, chamados de tipo I e tipo II, que expressam marcadores moleculares diferentes e exercem funções distintas 19 , 24 , 25 . Bioquimicamente, os pericitos do tipo I são NG2 + Nestin - , enquanto os pericídios do tipo II são NG2 + Nestin + 19 , 24 . Funcionalmente, os pericitos tipo I podem sofrer diferenciação adipogênica, contribuindo para acúmulo de gordura e / ou fibrose, enquanto que os pericitos de tipo II podem se diferenciar ao longo da via miogênica, contribuindo para a regeneração muscular 19 , 24 , 25 . Estes resultados demonstram que: (1) os pericitos de tipo I podem bE alvo no tratamento de transtornos degenerativos gordos / fibrose, e (2) pericitos de tipo II têm grande potencial terapêutico para a distrofia muscular. Outras investigações e caracterização destas populações requerem um protocolo de isolamento que permita a separação dos pericitos tipo I e tipo II com alto grau de pureza. Atualmente, o isolamento de subpopulações pericíticas depende de camundongos duplamente transgênicos NG2-DsRed e Nestin-GFP 19,24. A disponibilidade de ratinhos NG2-DsRed ea qualidade da maioria dos anticorpos NG2 limitam o uso generalizado deste método. Dado que todos os pericitos NG2 + também expressam PDGFRβ nos músculos esqueléticos 19 , 20 , 24 , hipótese de que NG2 pode ser substituído por PDGFRβ para o isolamento de pericitos e suas subpopulações. Este trabalho descreve um protocolo baseado em FACS queUtiliza a coloração PDGFRβ e o sinal Nestin-GFP. Este método é menos exigente para os investigadores porque: (1) não requer o fundo NG2-DsRed e (2) utiliza anticorpos PDGFRβ comercialmente disponíveis, que são bem caracterizados. Além disso, permite o isolamento simultâneo de pericitos tipo I e tipo II com elevada pureza, permitindo uma investigação mais aprofundada da biologia e potencial terapêutico destas subpopulações de pericitos. Após purificação, estas células podem ser cultivadas em cultura, e as suas morfologias podem ser visualizadas. Este trabalho também mostra que os pericitos tipo I e tipo II isolados usando este método, como os purificados a partir de camundongos duplamente transgênicos, são adipogênicos e miogênicos, respectivamente.

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isolation of type i and type ii pericytes from mouse skeletal muscles

Pericitos são células multipotentes perivasculares que mostram heterogeneidade em diferentes órgãos ou mesmo dentro do mesmo tecido. Nos músculos esqueléticos, há pelo menos duas subpopulações de pericitos (chamadas de tipo I e tipo II), que expressam diferentes marcadores moleculares e possuem distintas capacidades de diferenciação. Utilizando ratinhos duplamente transgénicos NG2-DsRed e Nestin-GFP, os pericitos de tipo I (NG2-DsRed + Nestin-GFP-) e de tipo II (NG2-DsRed + Nestin-GFP + ) foram isolados com sucesso. Contudo, a disponibilidade destes ratinhos duplamente transgénicos impede o uso generalizado deste método de purificação. Este trabalho descreve um protocolo alternativo que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II a partir de músculos esqueléticos. Este protocolo utiliza a técnica de triagem de células activada por fluorescência (FACS) e alvos PDGFRβ, em vez de NG2, juntamente com o sinal de Nestin-GFP. Após o isolamento, o tipo I e tyOs pericitos II apresentam morfologias distintas. Adicionalmente, os pericitos de tipo I e de tipo II isolados com este novo método, como os isolados a partir de ratinhos duplamente transgénicos, são adipogénicos e miogénicos, respectivamente. Esses resultados sugerem que este protocolo pode ser utilizado para isolar subpopulações de pericitos dos músculos esqueléticos e, possivelmente, de outros tecidos.

Os parâmetros FACS, incluindo a intensidade do laser e a compensação do canal, são corrigidos com base nos resultados de controles sem cor e controles de cor única. O controlo PDGFRβ-PE-FMO é utilizado para definir o gating para a população de PDGFRβ-PE + ( Figura 1A ). Entre as células PDGFRβ-PE, duas populações que representam as células Nestin-GFP + e Nestin-GFP estão claramente separadas ( Figura...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Este trabalho descreve um protocolo baseado em FACS que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II dos músculos esqueléticos.

Isolamento de Pericitos Tipo I e Tipo II de Músculos Esqueléticos de Rato

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.1K visualizações.  University of Minnesota.  Este trabalho descreve um protocolo baseado em FACS que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II dos músculos esqueléticos. 

Vídeo: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolamento e Quantitative Immunocytochemical caracterização de células Miogênica primários e fibroblastos de músculo-esquelético humano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Biologia do Desenvolvimento

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caracterização de células-satélite de músculo de rato Cabeça branquiométrico

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Usando mitocôndrias isoladas de quantidades mínimas de Rato Músculo Esquelético para alta taxa de transferência de microplacas respiratórias Medições

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Isolamento eficaz de ilhotas funcionais a partir de ratinho neonato Pâncreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

Isolamento e Enriquecimento de fígado progenitoras Subconjuntos identificado por um marcador combinação de superfícies Novel

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Geração de padronizada e reprodutível Forebrain-tipo Organoids Cerebral de humanos induzida por células-tronco pluripotentes

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Dissecação da Retina Pupal drosófila para imuno-histoquímica, análise ocidental e isolamento de RNA

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Isolamento, cultura, caracterização e diferenciação de células progenitoras do músculo humano do procedimento de biópsia do músculo esquelético

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e diferenciação de Mioblasts primários de explantes do músculo esquelético do rato

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Traduzindo a Purificação da Afinidade ribossófica (TRAP) para investigar o desenvolvimento raiz arabidopsis thaliana em uma escala específica de tipo de célula

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...