Anyaporn Sawasdichai e Nazefah Abdul Hamid são gratos ao Governo Real da Tailândia e do Governo da Malásia, respectivamente, para doutorado Bolsas de estudo. Este trabalho foi financiado pelo Wellcome Trust um projeto de subvenção concedida ao PSJ e KG. Agradecemos também à Universidade de Bristol Facility BioImaging Wolfson.

I. Preparação para o fracionamento Esta seção descreve a preparação de poli-L-Lisina lamínulas de microscópio revestidas eo anexo de células antes do fracionamento. Se necessário as células podem ser transitoriamente transfectadas com vetores de expressão de proteínas antes ou depois de apego. A. Preparação de poli-L-Lisina lamínulas revestido <ol><li> Prepare uma solução de 1mg/ml poli-L-Lisina em água destilada. </li><li> Lamínulas revestimento limpo com poli-L-Lisina, incubando-as na solução por pelo menos uma hora em uma plataforma de balanço a 22 ° C. </li><li> Lavar as lamelas revestido com água destilada estéril por duas vezes e seguir com uma única lavagem com etanol 96%. </li><li> Ar seco as lamelas revestido em um pedaço de papel de filtro e mantê-los em um recipiente seco para uso futuro (uma vez seca, eles podem ser empilhados). </li></ol> Células B. Colocar em lamínulas Células não aderentes (aqui usamos células K562) <ol><li> Coloque uma lamela poli-L-Lisina revestido em um poço em um prato 6-bem com a superfície revestida voltada para cima. </li><li> Semente K562 células a uma densidade de 7x10 5 células / poço em Modified Dulbecco Eagles Medium (DMEM) suplementado com 10% soro fetal bovino (FBS) e PS (penicilina 100units/ml, estreptomicina 100mg/ml). No caso de células K562 transfecção transiente pode ser realizada utilizando eletroporação (0,4 centímetros cubetas de eletroporação com 1x10 7 células em 200μl dos meios de comunicação em 250V / 975μF) antes da semeadura das células. </li><li> Incubar as células por 24 horas a 37 ° C em 5% CO 2. </li><li> Deitar fora o meio e lave as células duas vezes com fosfato gelada solução salina tamponada (PBS). </li><li> Seguir com o protocolo de fracionamento subcelular. </li></ol> Células aderentes (aqui usamos Cos-7 células) <ol><li> Coloque uma lamela sem revestimento em um poço em uma placa de 6 poços. </li><li> Lavar duas vezes em PBS pré-aquecido a 37 ° C. </li><li> Separar as células por digestão com tripsina (0,03% EDTA, 0,25% de tripsina) em PBS a 37 ° C por 2-5 minutos. É importante não para mais de digerir as células e para parar a digestão, assim como predominantemente as células individuais flutuante estão presentes. </li><li> Parar a digestão semeando as células a uma densidade de 3x10 5 células / poço em uma placa de 6 poços em DMEM suplementado com 10% FBS e PSG (penicilina 100 unidades / ml, estreptomicina 100ug/ml e L-glutamina 292ug/ml) , em lamínulas normal. Células aderentes geralmente atribuem bem em lamínulas de microscópio normal, no entanto, poli-L-Lisina lamínulas revestido pode ser usado se for necessário. </li><li> Incubar as células por 24 horas a 37 ° C e 5% CO 2. No caso da Cos-7 células transfecção transiente pode ser realizada utilizando Fugene 6 (Roche) se necessário, e as células da esquerda para a recuperação por mais 24 horas a 37 ° C e 5% de CO 2. </li><li> Deitar fora o meio e lave as células duas vezes com PBS gelado. </li><li> Seguir com o protocolo de fracionamento subcelular. </li></ol> II. Fracionamento subcelular Na fração situ é realizada de acordo com o esquema mostrado na Figura 1 e é baseado em um método descrito anteriormente 5, com modificações descritas anteriormente 7 e no protocolo detalhado abaixo. É recomendado para transferir as lamelas de um prato 6-bem fresco após cada etapa de fracionamento de remover a lamínula antes de retirar o líquido. Embora apenas quatro lamelas são necessários para imagem, lamínulas adicionais são necessários para produzir extrato celular total e frações matriz nuclear para western blotting se isso é para ser realizado em paralelo. <ol><li> Preparar e filtro citoesqueleto buffer (CSK): TUBOS 10mM, sacarose 300mM, NaCl 100mM, MgCl 3mM 2, 1 mM EGTA. CSK tampão deve ser preparada no dia do fracionamento. </li><li> Se necessário, a células de uma lamela pode ser usado para preparar um extracto de células inteiras de western blotting colocando delicadamente tampão 200ul TES (1% SDS, 2mM EDTA, 20mM Tris-HCl pH 7,4) diretamente sobre a lamela e incubar durante 1 ou 2 minutos a 22 ° C. O extrato de células inteiras podem então ser removidos e as proteínas precipitadas por adição de 250ul de 1M (NH 4) 2 SO 4 e incubar no gelo até a outras amostras ocidentais estão prontos. </li><li> Lavar as lamelas restantes com 1 ml gelada PBS duas vezes a 22 ° C pela inclinação do 6 bem-placa duas ou três vezes. </li><li> Retirar as lamelas representando células inteiras e transfira para um prato 6-bem fresco. Siga com a fixação das células e imuno-fluorescência protocolo. </li><li> Remova cuidadosamente a PBS dos poços restantes. </li><li> Extrair as proteínas citoplasmáticas e vagamente realizada nuclear colocando delicadamente tampão 200ul CSK + 0,1% (V / V) Triton X-100 diretamente em cada lamela restantes e incubação emgelo por 1 minuto. </li><li> Retirada do extrato citoplasmático e vagamente realizada nuclear e precipitar como descrito na etapa 2. </li><li> Lavar as lamelas com gelo 1ml frio PBS duas vezes a 22 ° C pela inclinação do 6 bem-placa. </li><li> Retirar as lamelas representando bem realizada material nuclear e siga com a fixação das células e imuno-fluorescência protocolo. </li><li> Remova cuidadosamente a PBS dos poços restantes em seguida, extrair as proteínas são mantidas em segredo colocando delicadamente tampão 200ul CSK + 0,5% (V / V) Triton X-100 diretamente em cada lamela restantes e incubar no gelo por 20 minutos. </li><li> Retirada do extrato são mantidas em segredo e precipitar como descrito na etapa 2. </li><li> Lavar as lamelas com gelo 1ml frio PBS duas vezes a 22 ° C pela inclinação do 6 bem-placa. </li><li> Retirar as lamelas representando fração cromatina e siga com a fixação das células e imuno-fluorescência protocolo. </li><li> Remova cuidadosamente a PBS dos poços restantes em seguida, coloque 200ul CSK tampão + 100ug/ml de DNase I para as lamelas restantes e incubar por 30 minutos a 37 ° C. </li><li> Retirada do extrato da cromatina e precipitar como descrito na etapa 2. </li><li> Lavar as lamelas com gelo frio PBS duas vezes a 22 ° C pela inclinação do 6 bem-placa. </li><li> Retirar as lamelas que representa a fração da matriz nuclear e siga com a fixação das células e imuno-fluorescência protocolo. </li><li> Se necessário, a proteínas da matriz pode ser removido de uma lamela matriz adicionais para western blotting utilizando tampão TES 200ul como descrito na etapa 2. </li><li> Amostras para western blot deve ser centrifugado a 13.000 rpm em microcentrífuga de bancada a 4 ° C e os sedimentos ressuspensos em 40ul SDS loading buffer (62,5 mm Tris-HCl pH 6,8, SDS 2% (w / v), 50 mM DTT, glicerol 10%, 0,01% de azul de bromofenol (w / v)) antes da análise por SDS-PAGE. <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Figura 1" /> Figura 1. A representação esquemática de no fracionamento subcelular situ. Citoesqueleto buffer (CSK) consiste em PIPES 10mM, sacarose 300mM, NaCl 100mM, MgCl 3mM 2, 1 mM EGTA. TES buffer consiste de 1% SDS, 2mM EDTA, 20mM Tris-HCl pH 7.4. </li></ol> III. Fixação das células e imuno-fluorescência <ol><li> Delicadamente adicionar 1 ml de formaldeído a 4% / PBS a cada lamela e incubar a 4 ° C por 30 minutos. </li><li> Lave cada lamela suavemente 2-3 vezes com 1ml de PBS a 22 ° C. </li><li> Adicionar 400ul de X-100/PBS Triton 0,1% a cada lamela e incubar a 22 ° C por 10 minutos. </li><li> Lave cada lamela suavemente 2-3 vezes com 1ml de PBS a 22 ° C. </li><li> Adicionar 400ul de 3% albumina sérica bovina (BSA) / PBS e incubar a 22 ° C por 20 minutos para reduzir a coloração de fundo em potencial. </li><li> Lave cada lamela suavemente 2-3 vezes com 1ml de PBS a 22 ° C. </li><li> 100ul lugar do anticorpo primário em PBS diretamente sobre a lamela e incubar a 22 ° C por 1 hora. Anticorpos primário e secundário deve ser diluído em PBS a concentração necessária. Este pode ter de ser otimizado para cada anticorpo utilizado. </li><li> Lave cada lamela suavemente 2-3 vezes com 1ml de PBS a 22 ° C. </li><li> 200ul local de anticorpo secundário em PBS e incubar a 22 ° C ao abrigo da luz por 1 hora. </li><li> Lave cada lamela suavemente 2-3 vezes com 1ml de PBS a 22 ° C. </li><li> Monte cada lamela (células para baixo) sobre uma lâmina de vidro usando Vectashield meio de montagem contendo DAPI (4 &#39;,6-diamidino-2-phenylindole de cloridrato). </li><li> Limpe o excesso de meio de montagem de todo o lamela usando uma toalha de papel e incubar a lâmina a 22 ° C por pelo menos 30 minutos de distância da luz. </li><li> Fixar a lamínula para a lâmina de vidro utilizando unha polonês acrílico transparente. </li><li> Manter os slides longe da luz antes de visualização por microscopia de fluorescência. </li></ol> IV. Resultados representativos <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Figura 2" /> Figura 2. Fracionamento subcelular de não-transfectadas COS-7 células. Fracionado Cos-7 células foram fixadas com formaldeído e imunocoradas usando α-tubulina, α-histona H1 ou α-Lamin A anticorpos / C conjugada com TRITC seguido de tratamento com meio de montagem contendo DAPI . Imagens foram adquiridas com lente ocular de 10x e lente objetiva 63x usando um microscópio confocal Leica. Núcleos celulares foram visualizadas através de um conjunto de filtros DAPI, enquanto no mesmo campo células coradas com anticorpos marcador foram visualizadas através de um conjunto TRITC filtro. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Figura 3" /> Figura 3. A proteína de fusão GFP-6E2 é associado com a matriz nuclear. Fracionamento subcelular de Cos-7 células que expressam uma proteína de fusão composta de proteína verde fluorescente (GFP) fundida ao papilomavírus humano (HPV) do tipo 6E2 proteína (GFP-6E2). Núcleos celulares foram visualizadas através de coloração DAPI, enquanto localização de GFP-6E2 nas células transfectadas foi diretamente visualizadas através GFP fluorescência. Lamin A / C foi visualizado utilizando α-Lamin A anticorpos / C conjugada com TRITC e um conjunto TRITC filtro. As imagens revelam que alguns fusão da proteína GFP-6E2 é associado com a matriz nuclear (painel inferior direito). As imagens foram obtidas como descrito na Figura 2.

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

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Problemas comuns e sugestões: Todos ou a maioria das células são perdidas durante a lavagem. Durante as etapas de líquidos de lavagem deve ser adicionado lentamente para o lado do prato 6-bem evitando a lamela. Da mesma forma os líquidos devem ser retirados com cuidado a placa de inclinação e, lentamente, pipetagem off excesso de líquido. Adesão celular pode ser aumentada usando poli-L-Lisina lamínulas revestido. DNA genômico não é completamente digerid...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

Função da proteína está intimamente acoplado a localização de proteínas. Apesar de algumas proteínas são restritos a um local específico ou compartimento subcelular, muitas proteínas estão presentes como uma população livre difusão de livre troca com uma sub-população que está fortemente associado a um domínio particular subcelular ou estrutura. Fracionamento subcelular Em situ permite a visualização de compartimentalização de proteína e pode também revelar proteína sub-populações que localizar a estruturas específicas. Por exemplo, a remoção de proteínas solúveis citoplasmáticas e vagamente realizada proteínas nucleares pode revelar a associação estável de alguns fatores de transcrição com cromatina. Digestão subseqüente de DNA pode, em alguns casos revelar associação com a rede de proteínas e RNAs que é coletivamente chamado de andaime nuclear ou matriz nuclear. Aqui nós descrevemos os passos necessários durante o fracionamento em situ de aderentes e não aderentes células de mamíferos em lamínulas de microscópio. Visualização de proteínas pode ser conseguido usando anticorpos específicos ou proteínas de fusão fluorescentes e microscopia de fluorescência. Anticorpos e / ou corantes fluorescentes que funcionam como marcadores para compartimentos específicos ou estruturas permitem localização de proteínas a ser mapeada em detalhes. No fracionamento situ também pode ser combinado com western blotting para comparar as quantidades de proteínas presentes em cada fração. Esta abordagem simples bioquímicos podem revelar associações que poderiam passar despercebido.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

No fracionamento subcelular situ de células de mamíferos em lamínulas de microscópio permite a visualização de localização de proteínas.

Fracionamento em Subcelulares situ de aderentes e não aderentes células de mamíferos

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K visualizações.  University of Bristol. No fracionamento subcelular situ de células de mamíferos em lamínulas de microscópio permite a visualização de localização de proteínas.

Vídeo: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Subcultura células de mamíferos

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Biologia

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSÃO para a seleção RNAi em células de mamíferos

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogênica Ensaio: células aderentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análise da posição do ciclo celular em células de mamíferos

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualização de mRNAs retículo endoplasmático localizados em células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polysome Fracionamento e Análise de mamíferos Translatomes em uma escala genômica

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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 Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...