Trabalho descrito neste manuscrito foi possível graças ao financiamento da Fundação Patrick F. Taylor.

1. Cromossomo colheita de células aderentes <ol><li> Protocolo padrão <ol><li> Cultivar células de acordo com as condições específicas de cultivo celular. Quando as células atingiram fase logarítmica (80% de confluência), adicionar 10 ul / ml de Colcemid com o frasco de cultura de célula. Recomenda-se um período mínimo de 2 x 10 6 células. </li><li> Incubar as células a 37 ° C numa atmosfera de 5% de CO2 durante 45 min. Usando uma pipeta estéril, transferir mídia a partir de células em um tubo de 15 ml. Ponha de lado. </li><li> Lavar suavemente as células por adição de 2 ml de tampão de HBSS para o balão. Tampão do redemoinho e, em seguida, retire com uma pipeta. Descarte. </li><li> Adicionar 1 ml de tripsina, assegurando que cobre toda a superfície do frasco. Apenas deixar as células em tripsina durante cerca de 2 min. Uma vez que a maioria das células têm destacado, pipetar os meios de comunicação do tubo cônico de volta para as células. </li><li> Transferência da suspensão de células em 10 ml de partes alíquotas em tubos de 15 mL cónicos.Centrifugar a 200 xg durante 10 min. Remover o sobrenadante e ressuspender o pellet. </li><li> Adicionar 10 ml de 0,075 M de KCl, que foi pré-aquecido a 37 ° C e a pelete remanescente no tubo cónico. Tubo Vortex em velocidade média de misturar KCl e células. </li><li> Incubar as células a 37 ° C durante 10 min. Centrifugar a 200 xg durante 5 min a 25 ° C. Remover o sobrenadante (até cerca de 0,5 ml restos) e ressuspender pellet. </li><li> Adiciona-se cuidadosamente 5 ml de fixador de Carnoy fresco (proporção de 3:1 de metanol: ácido acético glacial) para as células, enquanto a vórtex. Em seguida, adicionar 5 ml de fixador mais sem vortex para um total de 10 ml. </li><li> Centrifugar a 200 xg durante 5 min. Remover as células sobrenadante e ressuspender. Adicionar 5 ml de fixador para cada tubo. </li><li> Centrifugar a 200 xg durante 5 min. Remover as células sobrenadante e ressuspender. Adicionar 5 ml de fixador para cada tubo. As células podem agora ser armazenados em 4 ° C, durante até um ano. </li></ol></li><li> Modificação de Protocolo: harvesti cromossômicasng de linhas de células linfoblastóides <ol><li> Cultivar células de acordo com as condições específicas de cultivo celular. (Use o frasco adequado para a quantidade de células em cultura). Quando as células atingiram fase logarítmica (cerca de 2 dias após as células são de divisão), adicionar 10 ul / ml de Colcemid com o frasco de cultura de célula. </li></ol></li><li> Modificação de Protocolo: colheita cromossomo do sangue total Fito-hemaglutinina (PHA), uma lectina a partir de derivados do feijão vermelho, é um mitogénio potente para as células T humanas 16. 72 horas após a adição da PHA para a cultura, a cerca de 45% das células estão em fase S. Isto representa o pico de atividade mitótica, e é o ponto ideal em que a colheita para os estudos cromossômicos. Outros mitogenos como pokeweed (1-10 ug / ml) pode ser usado na análise de células B 17,18. <ol><li> Coletar pelo menos 1 ml de sangue total em um tubo de heparina de sódio topo verde. Use dentro de 3 dias após a colheita. Guarde-o em sangue RT until pronto para usar. </li><li> Aliquota de 0,25 mL de sangue total em 10 ml de meio RPMI completo contendo L-glutamina (20% de soro fetal bovino, 1% de penicilina / estreptomicina, 1% de fungizona, e 1% de PHA). Cultura a 37 ° C com 5% de CO 2. </li></ol></li><li> Modificação de Protocolo: colheita cromossomo da medula óssea A análise citogenética de medula óssea é útil para muitas doenças hematológicas malignas como a observação de um clone cromossomicamente anormal podem constituir um diagnóstico para um tipo específico de leucemia. Estudos cromossómicas também são utilizados para avaliar a progressão da doença, tais como o aparecimento de crise explosão e resposta ao tratamento. Como as células da medula óssea estão dividindo ativamente, nenhum estímulo mitógeno é necessário 9,19. Para leucemias agudas 24 e 48 culturas de RH também são criados. <ol><li> Coletar pelo menos 1 ml de medula óssea em um tubo de heparina de sódio topo verde. Aliquota de 0,25 ml de medula óssea em 10 ml de meio RPMI completo contendo L-glutamina (20% de soro fetal bovino, 1% de penicilina / estreptomicina, 1% de fungizona). Cultura a 37 ° C com 5% de CO 2. </li></ol></li></ol> 2. Deslize Preparação e coloração sólida Uma avaliação rápida de um slide representante irá fornecer informações sobre a qualidade da colheita, antes de continuar a novos procedimentos, tais como G-bandas, FISH, CGH, ou SKY. Alguns laboratórios preferem mancha sólida das células para a colheita rápida e avaliação cromossômica. Alternativamente, um microscópio de contraste de fase pode ser usado para esta análise. Os slides são mais bem preparados quando a umidade é de aproximadamente 50% ea temperatura ambiente (20-25 ° C). <ol><li> Centrifugar as células a 200 xg durante 5 min a 25 ° C. Remover o sobrenadante até que apenas 0,3-0,5 ml restos mortais. </li><li> Após ressuspensão da pelota, suavemente as pipetas três gotas da suspensão de células a partir de uma distância de cerca de 2 em sobre uma lâmina que é inclinada num ângulo de cerca de 45 ° e permite que a suspensãorolar em todo o slide. Adicione uma grande queda de Fixação fresco de Carnoy ao slide. </li><li> Seque a parte de trás do slide em uma toalha de papel e, em seguida, sentar-se a deslizar para fora para secar por pelo menos 10 min. O slide deve estar completamente seca. </li><li> Prepare fresco Giemsa solução corante (3:1 relação de Gurr buffer e Giemsa). Colocar as lâminas em um rack de coloração. Cubra todo o slide na solução de Giemsa. Deixe as lâminas permanecem na solução de coloração, durante 5 min. Lavar as lâminas com água destilada, escorra e deixe secar ao ar. </li><li> Adicionar 4 gotas de Permount e uma lamela para o slide. Certifique-se de que não há bolhas sob a lamela. O excesso Permount pode ser removida com uma toalha de papel. </li><li> Analise as células com um microscópio de luz em 10X e 100X de ampliação. Se as células em metafase são abundantes e bem distribuídos, os restantes lâminas pode ser utilizado para outros procedimentos experimentais. </li></ol> 3. G-Banding Usando tripsina e Giemsa (GTG) &lt;p&gt; A tripsina é uma enzima proteolítica que desnatura histonas eucromáticos em regiões de ADN com actividade de transcrição mais elevado resultante. Após a coloração de Giemsa, estas regiões aparece como bandas claras. Cromatina altamente condensada com atividade transcricional pouca ou nenhuma (heterocromatina) terá uma grande parte de seus histonas protegidos da tripsina e, portanto, vai manchar sombriamente após coloração Giemsa. É essencial inicialmente G-banda de um slide para monitorar as condições e ajustar o tempo de tripsina se necessário, para os slides seguintes. <ol><li> Adicionar as seguintes soluções para 4 frascos Coplin. Frasco # 1 = 30 mL de 1x de HBSS e 4 ml de 10x de tripsina (0,5%) Jar # 2 = 50 ml de HBSS 1x Frasco # 3 = 45 mL de HBSS e 1x 5 ml de soro fetal bovino Jar # 4 = 50 ml de HBSS 1x </li><li> Mergulhe cada slide no frasco n º 1 por 5 segundos, lavagem rápida no frasco n º 2, deixe cada slide no frasco n º 3, durante pelo menos 30 segundos, lavagem rápida no frasco n º 4, em seguida, deixar as lâminas secar. </li><li> Prepare fresh Giemsa solução corante (3:1 relação de Gurr buffer e Giemsa). Colocar as lâminas em um rack de coloração. Cubra todo o slide na solução de Giemsa. Deixe as lâminas permanecem na solução de coloração, durante 5 min. </li><li> Lavar as lâminas em água destilada na mesma ordem em que foram coradas. Permitir que as lâminas para secar por cerca de 10 min. O slide deve estar completamente seca. </li><li> Adicionar 4 gotas de Permount e uma lamela para o slide. Certifique-se de que não há bolhas sob a lamela. O excesso Permount pode ser removida com uma toalha de papel. </li><li> Analise as células com um microscópio de luz em 10X e 100X de ampliação. Ajustar tripsina temporização do resto das lâminas, com base nos resultados da primeira lâmina. </li></ol>

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Utilizámos o presente procedimento para o isolamento do cromossoma a partir de células de vários organismos, incluindo diferentes tipos de células obtidas a partir de humanos, macacos Rhesus, ratinhos e ratos, 11,20-22. O protocolo padrão é fornecida, mas certas etapas principais variáveis ​​e podem ter de ser ajustados para estes diversos tipos de células. Vários passos específicos são cruciais tanto na preparação cromossômica e G-faixas para garantir a melhor qualidade possível dos result...

Análise cromossômica é uma técnica convencional utilizada em todo o mundo para diagnosticar instabilidade cromossômica e rearranjos que levam a desordens genéticas e malignidade 1,2,8,9. Além disso, uma maior resolução para o diagnóstico e pesquisa de anormalidades genéticas e adquiridas constitucionais-cancro pode ser conseguido com a combinação dos procedimentos citogenéticas clássicas e metodologias citogenética molecular, tais como hibridação in situ fluorescente (FISH), a hibridação genómica comparativa (CGH) e cariotipagem espectral (SKY) 14,15. Mais recentemente, estas técnicas têm sido utilizadas para a avaliação de instabilidade cromossoma associado com células estaminais. Anormalidades cariotípico tais como aneuploidia de células de longo prazo cultivadas embrionárias (ES) e células estaminais adultas de vários organismos têm sido relatados por vários laboratórios. Evidências recentes comprovam que algumas linhas de células são inerentemente mais inclinados ao cromossomo instabidade, independentemente das condições de cultivo. Por esta razão, ao estabelecer e / ou manutenção de linhas de células estaminais Rhesus humano, rato, ou, análise cromossômica é recomendado como parte do processo de controle de qualidade. Muitos relatórios descrevem o aumento do interesse na utilização de rotina e citogenética molecular para monitorar a estabilidade cromossómico de células estaminais e células malignas ou vários organismos em cultura 10-13. Estes protocolos são cada vez mais utilizados por laboratórios não genéticos para avaliação cromossômica rápida de suas células cultivadas 13. Nós apresentamos os procedimentos básicos para a preparação do cromossoma a partir de vários tipos de células, as quais podem ser aplicadas para ambos os fins e as células clínicos e de pesquisa provenientes de vários organismos.

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

Análise cromossómica (citogenética) é amplamente utilizado para a detecção de instabilidade cromossoma. Quando seguido por G-faixas e técnicas moleculares como a hibridização fluorescente in situ (FISH), este ensaio tem a poderosa capacidade de analisar células individuais para as aberrações que envolvem ganhos ou perdas de partes do genoma e rearranjos envolvendo um ou mais cromossomos. Nos seres humanos, anormalidades cromossômicas ocorrem em cerca de 1 a cada 160 nascidos vivos 1,2, 60-80% de todos os abortos espontâneos 3,4, 10% dos natimortos 2,5, 13% dos indivíduos com doença cardíaca congênita 6, 3-6% dos casos de infertilidade 2, e em muitos pacientes com atraso no desenvolvimento e defeitos de nascimento 7. A análise citogenética de malignidade é rotineiramente utilizado por pesquisadores e clínicos, como observações de anormalidades cromossômicas clonais foram mostrados para ter tanto significado diagnóstico e prognóstico 8,9.60; isolamento cromossomo é de valor inestimável para a terapia gênica e células-tronco de organismos, incluindo primatas não-humanos e roedores 10-13. Os cromossomas podem ser isolados a partir de células de tecidos vivos, incluindo os linfócitos, fibroblastos da pele, amniócitos, placenta, da medula óssea e espécimes de tumor. Os cromossomas são analisados ​​na fase metafase da mitose, quando elas são mais condensada e, por conseguinte, mais claramente visíveis. O primeiro passo da técnica de isolamento cromossoma envolve o rompimento das fibras do fuso por incubação com Colcemida, para evitar que as células de prosseguir para a fase subsequente anafase. As células são, então, tratada com uma solução hipotónica e conservados no seu estado inchado com fixador de Carnoy. As células são, então, deixada cair sobre as lâminas e pode então ser utilizado para uma variedade de procedimentos. G-bandas envolve o tratamento de tripsina seguida de coloração com Giemsa para criar luz característica e faixas escuras. O mesmo procedure para isolar os cromossomas podem ser utilizados para a preparação de células para os procedimentos, tais como hibridação in situ fluorescente (FISH), a hibridação genómica comparativa (CGH) e cariotipagem espectral (CÉU) 14,15.

Metáfase alta qualidade são essenciais para a análise cromossômica. Um ensaio bem sucedido produz cromossomos, que são bem espalhados e da morfologia cromossômica adequado. Cromossomos corretamente G-unido conter a luz característica e padrões de bandas escuras. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> Figura 1. Uma propagação cro...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

Os cromossomas podem ser isolados a partir de células vivas, tais como linfócitos ou fibroblastos de pele, e a partir de organismos, incluindo seres humanos ou ratinhos. Estas preparações cromossômicas pode ser ainda utilizado para a rotina de bandamento G e procedimentos de citogenética molecular, como a hibridização fluorescente in situ (FISH), hibridização genômica comparativa (CGH), e cariotipagem espectral (SKY).

Cromossomo Preparação a partir de células cultivadas

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.2K visualizações.  Louisiana State University Health Science Center.  Os cromossomas podem ser isolados a partir de células vivas, tais como linfócitos ou fibroblastos de pele, e a partir de organismos, incluindo seres humanos ou ratinhos. Estas preparações cromossômicas pode ser ainda utilizado para a rotina de bandamento G e procedimentos de citogenética molecular, como a hibridização fluorescente in situ (FISH), hibridização genômica comparativa (CGH), e cariotipagem espectral (SKY). 

Vídeo: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Contagem e determinação da viabilidade das células em cultura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Biologia

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparação de Abóboras Drosophila cromossomos politênicos para Rotulagem de anticorpos

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualização do retículo endoplasmático Subdomains em células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Ensaios Repórter Massively Parallel em células de mamíferos cultivadas

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

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 ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Um cromossomo Dropping Ar seco rápido Método de Preparação Adequado para FISH em plantas

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparação de cromossomos se espalha a partir do Mouse espermatócitos

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Co-imunoprecipitação ensaio usando proteínas nucleares endógenas de células cultivadas em condições de hipóxicos

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

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

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