Os autores agradecem o aconselhamento e apoio técnico fornecido pelo Dr. Richard Konz e pessoal nas instalações Núcleo Citometria de Fluxo na Universidade de Massachusetts Medical School. Recursos suportados pelo núcleo de Endocrinologia Diabetes Research Center Grant DK032520 também foram utilizados. Este trabalho é suportado por concessões do National Institute of Mental Health (NIMH), do Instituto Nacional de Abuso de Drogas (NIDA) e do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano.

Extração de núcleos <ol><li> Colocar 250 mg de tecido congelado cérebro pós-morte em 5 mL de Lise um tampão em um douncer e colocá-lo no gelo. Para receber cerca de 5 milhões núcleos após FACS triagem, geralmente usamos 1000 mg de tecido por amostra. </li><li> Uma vez que o tecido tem descongelado, Dounce o tecido por 1 minuto enquanto no gelo. </li><li> Pegue o tecido homogeneizado e colocá-lo em um tubo de ultracentrífuga 15 mL claro. Colocar o tubo em gelo. </li><li> Tome 9 mL da solução de sacarose 2 com uma pipeta, coloque a pipeta no fundo do tubo de ultracentrífuga claro, e liberar a solução de sacarose 2 para criar um gradiente de concentração com a solução de tecido homogeneizado em cima da solução de sacarose 2. </li><li> Pesar os tubos de ultracentrífuga para se certificar de que todos eles vão ser equilibrada ao girar dentro da ultracentrífuga. Faça os ajustes para o peso pela adição de Tampão de Lise 1 para a camada superior. </li><li> Após os tubos foram pesadas, colocá-los em baldes do rotor. </li><li> Coloque todos os baldes em um rotor SW28 e coloque cuidadosamente o rotor para a ultracentrífuga. </li><li> Ultracentrífuga as amostras em 107.163,6 RCF por 2,5 horas a 4 ° C. </li><li> Uma vez que a centrifugação for concluída, remova o sobrenadante, juntamente com a camada de detritos encontrados no gradiente de concentração, com a utilização de um vácuo, tomando cuidado para não perturbar o pellet contendo os núcleos na parte inferior do tubo. </li><li> Adicionar 500 mL de 1X PBS a cada pellet e deixe descansar na geladeira por 20 minutos. Isso permite que o pellet para dissolver um pouco por conta própria, tornando mais fácil para depois dissolvê-lo mecanicamente por pipetagem cima e para baixo, o que diminui a quantidade de estresse a experiência dos núcleos. </li><li> Após os núcleos têm incubadas no gelo por 20 minutos, mecanicamente pipeta cima e para baixo para dissolver completamente os núcleos dentro 1XPBS. </li><li> Remover a solução contendo núcleos dos tubos de ultracentrifugação e combinar o conteúdo de dois tubos em um único tubo de microcentrífuga 2 mL. Novamente, coloque as amostras em gelo. </li></ol> Imunomarcação para FACS <ol><li> Neste ponto, faça uma mistura composta imunomarcação do anticorpo primário e secundário, e Bloqueio Mix. Para cada amostra, combine 300 mL de 1XPBS contendo 1,2 mL de anticorpo NeuN, 100 mL da mistura de bloqueio, que contém BSA 0,5% e soro de cabra normal em 10% 1XPBS, e 1 mL de Alexa Fluor 488. Para as amostras controle, excluir a NeuN anticorpo primário e, em vez disso, basta adicionar 1XPBS mais. </li><li> Vortex da mistura imunocoloração e incubar por 5 minutos em temperatura ambiente no escuro. </li><li> Enquanto a mistura de coloração é a incubação, fazer os controles para as amostras. FACS para triagem, uma amostra, contendo núcleos só, chamado de &quot;controle sem mácula&quot; é necessário, uma segunda amostra, um controle negativo, contendo os núcleos com o anticorpo secundário Alexa Fluor 488 sem o anticorpo primário NeuN também é necessário. </li><li> ML alíquota 20 dos núcleos contendo solução para um tubo de microcentrífuga contendo 2 mL 1XPBS para o controle sem manchas, e outro 20 mL para outro tubo de 2 ml contendo 980 uL de 1XPBS para o controle negativo. Aumentar o volume dos núcleos contendo amostra de volta até 1 mL com 1XPBS. Todas as amostras são colocadas de volta no gelo. </li><li> Agora que a mistura imunocoloração terminou incubação, adicione o seu conteúdo de 401 mL em amostras adequadas. A amostra núcleos receberão a mistura contendo ambos os NeuN e Alexa Fluor 488, enquanto a amostra negativa receberá a mistura contendo apenas o anticorpo secundário Alexa Fluor 488. </li><li> Tomar as amostras para a sala fria para rodar no escuro por 45 minutos. </li><li> Depois que as amostras têm incubadas no escuro por 45 minutos, colocá-los no gelo e trazê-los para a instalação FACS. Durante o transporte para a instalação FACS, as amostras devem ser mantidas no gelo e no escuro. </li></ol> FACS <ol><li> Na instalação FACS, filtro as amostras através de um filtro de 40 um. Então, executá-los através do instrumento por alguns minutos, enquanto sendo mantido frio, a fim de obter alguns dados preliminares antes da separação. </li><li> Neste ponto, é necessário definir as portas adequadas para a classificação. Várias portas diferentes são usados ​​para amostras tipo. O primeiro portão dá uma idéia do tamanho dos núcleos diferentes encontrados dentro da amostra, e permite separatation de detritos de núcleos reais. O segundo portão nos permite classificar os núcleos individualmente. Descartar qualquer agregados são descartados neste momento. Finalmente, o terceiro portão é definido para um determinado comprimento de onda de fluorescência, correspondente ao do fluorocromo encontrada na nossa anticorpo secundário. Colocando esse portão que nos permite classificar e + NeuN separado, portanto núcleos neuronais de NeuN - núcleos. </li><li> Depois de todas as portas são escolhidos, os núcleos podem ser classificados em 1XPBS. </li><li> Uma vez que oamostra total passou, verificar a pureza da espécie, executando uma alíquota da amostra classificadas através do instrumento. Certifique-se que fluorescence da amostra não se espalha, mas é contido dentro de um único quadrante. É melhor escolher amostras que são mais de 90% pura. </li></ol> Depois FACS <ol><li> Uma vez que a amostra foi classificada, o processamento dos núcleos pode começar. Tomar 10 mL de amostra classificados e adicionar-lhe 2 mL da solução de sacarose 2, 50 uL de 1M CaCl 2 e 30 ul de 1m Mg (Ace) 2. Como um lembrete, certifique-se sempre manter a amostra em gelo desde os núcleos são corrigidos. </li><li> Se houver menos de 10 mL de amostra classificados, aumentar o volume até 10 mL, adicionando 1XPBS, ou ajustar a materiais adicionais em conformidade. </li><li> Inverter a amostra várias vezes e colocar no gelo por 15 minutos. </li><li> Após as amostras terem incubado em gelo por 15 minutos, centrifugar-los em um balde de rotor swing em 1786 RCF por 15 minutos a 4 ° C. </li><li> Desprezar o sobrenadante com o uso de um vácuo, sem perturbar o sedimento branco encontrado na parte inferior do tubo. </li><li> Dissolver o sedimento em 200 mL de qualquer solução que você precisa para os experimentos de prosseguir, e pipetar cima e para baixo. </li><li> Transferir a solução para um pequeno tubo e colocá-lo no freezer -80 ° C até à sua utilização. </li></ol>

<ol>
	<li>Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., Jaenisch, R., Laird, P. W., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+human+cerebral+cortex+is+dynamically+regulated+throughout+the+life+span+and+involves+differentiated+neurons.">DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.</a> PLoS ONE. 2, 895-895 (2007).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Acevedo, L. G., Iniguez, A. L., Holster, H. L., Zhang, X., Green, R., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+ChIP-chip+analysis+using+10,000+human+cells.">Genome-scale ChIP-chip analysis using 10,000 human cells.</a> Biotechniques. 43, 791-797 (2007).</li></ol>

O protocolo aqui apresentado deve ser particularmente útil para os estudos focados em mudanças epigenéticas da cromatina neuronal e, mais genericamente, sobre o fenótipo molecular do núcleo neuronal, durante a doença em desenvolvimento e envelhecimento, ou em condições normais neurológicos ou psiquiátricos. O método é uma vantagem particular quando a heterogeneidade celular de tecido do cérebro, incluindo mudanças potenciais no neurônio-glia de-ração durante o curso do envelhecimento ou devido a doenças são uma preocupaç...

Reagentes: <table><tr><th colspan="3" align="left"> 1. Tampão de Lise </th></tr><tr><td> 0.32M </td><td> Sacarose </td><td> 5,47 g </td></tr><tr><td> 5 mM </td><td> CaCl 2 </td><td> 250 L </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 mL </td></tr><tr><td> 0,1 mM </td><td> EDTA </td><td> 10 ml </td></tr><tr><td> 10mM </td><td> Tris-HCl, PH8 </td><td> 500 mL </td></tr><tr><td> 1 mM </td><td> TDT </td><td> 17 mL </td></tr><tr><td> 0,1% </td><td> Triton X-100 </td><td> 50 ul </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajustar o volume para 50 ml com água esterilizada </td></tr></table><table><tr><th colspan="3" align="left"> 2. Solução de sacarose </th></tr><tr><td> 1,8 M </td><td> Sacarose </td><td> 30,78 g </td></tr><tr><td> 3 mM </td><td> Mg (acetato) 2 </td><td> 150 mL </td></tr><tr><td> 1 mM </td><td> TDT </td><td> 17 mL </td></tr><tr><td> 10 mM </td><td> Tris-HCl, PH8 </td><td> 500 mL </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajustar o volume para 50 ml com água esterilizada </td></tr></table><table><tr><th colspan="3" align="left"> 3. Dounce tampão </th></tr><tr><td> 10 mM </td><td> Tris </td><td> 0,242 g </td></tr><tr><td> 4 mM </td><td> MgCl 2 </td><td> 0,163 g </td></tr><tr><td> 1 mM </td><td> CaCl 2 </td><td> 0,03 g </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> Ajustar o pH para 7,5 e volume para 200 ml com água esterilizada </td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

Neurônios no cérebro humano se tornar pós-mitóticos em grande parte durante o desenvolvimento pré-natal, e assim manter seus núcleos em todo o ciclo de vida completo. No entanto, pouco se sabe sobre as alterações na cromatina nuclear neuronal e na organização durante o curso do desenvolvimento e envelhecimento, ou doença neuropsiquiátrica crônica. No entanto, a data mais cromatina e DNA ensaios com base (que não FISH) a falta de resolução única célula. Para este fim, a uma considerável heterogeneidade celular de tecido cerebral representa uma limitação significativa, porque normalmente várias subpopulações de neurônios são misturados com diferentes tipos de células gliais e outras células não-neuronais. Uma possível solução seria a crescer células tipo culturas específicas, mas a maioria das células do SNC, incluindo os neurônios, são ex vivo sustentável, na melhor das hipóteses, por apenas algumas semanas e assim proporcionaria um modelo incompleto de mecanismos epigenéticos potencialmente operacional em todo o ciclo de vida completo . Aqui, nós fornecemos um protocolo para extrair e purificar a partir de núcleos congelados do cérebro pós-morte (nunca fixo) humana. O método envolve a extração de núcleos em tampão de lise hipotônica, seguido por ultracentrifugação e immunotagging com anti-NeuN anticorpos. Rotulados núcleos neuronais são então recolhidos separadamente usando fluorescência ativado classificação. Este método deve ser aplicável a qualquer região do cérebro em uma ampla gama de espécies e adequado para estudos de imunoprecipitação de cromatina com local e modificação específica anticorpos anti-histonas, e para a metilação do DNA e outros ensaios.

Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A heterogeneidade celular de tecido cerebral representa uma limitação significativa para o estudo de marcas epigenéticas na cromatina porque a maioria dos ensaios falta de resolução única célula. Neurônios são normalmente misturados com glia e outras células não-neuronais. Nós fornecemos um protocolo para extrair e coletar núcleos neuronal do cérebro humano.

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, ...

neuronal-nuclei-isolation-from-human-postmortem-brain-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.0K visualizações.  University of Massachusetts Medical School. A heterogeneidade celular de tecido cerebral representa uma limitação significativa para o estudo de marcas epigenéticas na cromatina porque a maioria dos ensaios falta de resolução única célula. Neurônios são normalmente misturados com glia e outras células não-neuronais. Nós fornecemos um protocolo para extrair e coletar núcleos neuronal do cérebro humano.

Vídeo: Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Um Ensaio Chromatin para tecido do cérebro humano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Biologia

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

Obtenção de RNA de alta qualidade a partir de populações de células no tecido humano único post-mortem do cérebro

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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 ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Isolamento à base de Afinidade de Tagged Núcleos de<em&gt; Drosophila</em&gt; Tecidos para Análise de Expressão Gênica

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolamento de ARN a partir de pequenas não-codificante de Soro Humano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento de RNA Rato Pâncreas: Um tecido rico em Ribonuclease

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 Enriquece o rendimento de melanócitos humanos de tecidos de pele adultos

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Isolamento de núcleos de tecido inteiro usando um método detergente e sem enzimas

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

Fluxo de trabalho de proteômica quantitativa usando detecção baseada em monitoramento de múltiplas reações de proteínas do tecido cerebral humano

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Isolamento Núcleos neuronais de Tecidos Humanos Postmortem Cérebro

Resumo

Explore mais vídeos

Capítulos neste vídeo