Danilo Maddalo é beneficiário de uma bolsa de pesquisa no Grupo de Jovem Pesquisador (YIG) do Instituto de Tecnologia de Karlsruhe.

1. Semeadura das células <ol><li> Antes de começar, aqueça o meio de cultura, a PBS 1X e da tripsina em banho-maria a 37 ˚ C </li><li> Tome as células fora da incubadora e aspirar o médio. </li><li> Aplicar suavemente 5 mL de PBS 1X sobre as células de lavá-las. </li><li> Aspirar o PBS 1X e aplicar 1 mL de tripsina contendo 0,025% EDTA. </li><li> Gire gentilmente a placa para ter uma distribuição uniforme da tripsina. </li><li> Coloque as células na incubadora a 37 ˚ C por 5-10 minutos (dependendo do tipo de célula). De tempos para verificar o tempo se as células são separadas por agitação da placa. </li><li> Ressuspender as células em 10-20 mL de meio e recolhê-las em um tubo falcon 50 mL. </li><li> Contar as células utilizando um contador Beckman. O número de células deve ser suficiente para garantir uma confluência de aproximadamente 60-70%. </li><li> Placa das células e permitir-lhes sementes de 6-8 horas. </li></ol> 2. Transfection <ol><li> Antes de transfecção, aquecer uma alíquota de soro livre médio em banho-maria a 37 ˚ C. </li><li> Trazer o Gene X-treme 9 a temperatura ambiente. </li><li> Pipeta o meio isento de soro em dois tubos de eppendorf (1 tubo de transfecção = 1). </li><li> Pipeta o Gene X-treme 9 na atenção meio isento de soro pagar que a ponta não toca a parede de plástico do eppendorf e incubar por 5 minutos. Nesse meio tempo preparar duas mixagens DNA: em um tubo de misturar os plasmídeos para renilla, vagalume e um controle do vetor vazio, em outro renilla tubo, firefly ea acompanhante molecular. </li><li> Adicione a mistura de DNA de cada eppendorf contendo meio livre de soro + X-treme Gene 9, vortex e incubar à temperatura ambiente por 20 minutos. </li><li> Adicionar gota a gota cada mistura em cada placa e incubar por 24 horas a 37 ° C e CO 2 5% </li></ol> 3. Divisão das células <ol><li> 24 horas após a transfecção, trypsinize as células como descrito em 1,4-1,6 e diluí-los a uma confluência final de 60-70% em uma placa de 6 poços. </li><li> Prepare um prato para o controle não-chocado e como muitos pratos como os pontos de tempo de recuperação que têm de ser analisados, como mostrado na figura. 2. </li></ol> 4. Choque térmico <ol><li> Para choque de calor, pré-aquecido soro contendo meio em banho-maria a 37 ˚ C. </li><li> &#39;Buffer de choque térmico &quot;preparar diluindo em meio cicloheximida soro contendo uma concentração final de 20 mcg / ml e MOPS a 20 mM. </li><li> Agora tire as placas da incubadora e aspire o meio. </li><li> Adicionar a cada 1 mL de bem &#39;buffer de choque de calor &quot;, e incubar por 30 minutos a 37 ˚ C e 5% de CO 2 (incluem também a placa de referência neste tratamento). Isto irá parar de novo a síntese de proteínas. </li><li> Enquanto isso no interruptor em um banho de água e definir o temperatura de 45 ˚ C. </li><li> Após a incubação, a tampa de vedação das placas com Parafilm previamente cortados em tiras. A placa de referência (100% de refolfed vagalume) fica na incubadora. </li><li> Incubar as placas a 45 ˚ C durante 30 minutos. </li><li> Remova as placas do banho-maria, retire o parafilme e colocar as placas de volta para a incubadora para permitir a recuperação. </li><li> Em cada ponto de colheita o tempo as células e lavá-los em PBS 1X por centrifugação em 2000Xg por 1 minuto a 4 ˚ C. </li></ol> 5. Lise celular e ensaio de luciferase <ol><li> Para uma lise celular eficiente, snap congelar o pellet celular em nitrogênio líquido. </li><li> Adicionar 200 mL de tampão de lise passiva diluído 1:5 em água destilada duas vezes em um tubo de vidro. </li><li> Adicionar 30 mL do lisado de células em uma placa de 96 também. Cada amostra é pipetados em triplicata. </li><li> Apenas o &quot;não chocado&quot; amostras de referência será utilizado paraler a atividade renilla luciferase, uma vez que esta etapa é utilizado para verificar a eficiência de transfecção iguais. </li><li> Preparar as soluções para o ensaio. Para a solução de luciferina luciferina diluir para uma concentração final de 0,2 mM em Gly-Gly buffer. Para o tampão de reação diluir TDT e ATP a concentração final 1mM em Gly-Gly buffer. Para tampão de reação celenterazina, diluir celenterazina para uma concentração final de 0,2 mM em tampão celenterazina. Mantenha solução luciferina e celenterazina luz tampão de reação protegida. </li><li> Agora coloque a placa de 96 poços na luminómetro e iniciar o programa &#39;Wallac 1420 Workstation. </li><li> Introduzir o Pump1 no tubo falcon contendo a solução celenterazina. Feche a tampa para que a luz não pode entrar em contato com o tubo. </li><li> Escolha no menu &#39;manutenção Dispenser&#39; a opção e marque Pump1. </li><li> Selecione a opção &quot;Fill&quot;. </li><li> Para editar o layout da placa escolha a opção &quot;Explorar protocolo e resultados&quot; no menu. Selecione o protocolo e clique duas vezes sobre ele. Agora selecione os poços a serem lidos. </li><li> Voltar ao &#39;Wallac Manager&#39; e clique em &quot;Iniciar&quot;. A reação entre o luciferase renilla e seu substrato celenterazina emite luz com um comprimento de onda de 482 nm. </li><li> Após a leitura, vá em &quot;manutenção Dispenser &#39;e selecione&#39; Vazio &#39;e Pump1 para liberar toda a solução residual de volta para o tubo. </li><li> Agora mova o tubo para um tubo falcon contendo água e marque a opção &#39;flush&#39;. </li><li> Após a etapa de lavagem, esvaziar o tubo por selecionar &#39;vazio&#39;. </li><li> Colocar o tubo de Pump1 no tubo do tampão de reação falcon e um da Pump2 no tubo falcon luciferina. </li><li> Alterar o layout da placa. </li><li> Selecione &#39;Preencher&#39; para Pump1 e Pump2 da Di &#39;menu de Spenser Manutenção &#39;. </li><li> Selecione o protocolo e iniciar a leitura. A reação entre o Firefly luciferase e luciferina seu substrato emite luz com um comprimento de onda de 560 nm. </li><li> No final da leitura repita o procedimento Vazio-Flush-vazio para Pump1 e Pump2. </li><li> Resultados já estão disponíveis no &#39;Wallac 1420 Explorer&#39;. </li><li> Cada experimento é associado a um número ensaio, nome de usuário, data de início e fim. </li><li> Resultados podem ser exportados como um arquivo de Excel. </li><li> Para os cálculos, as células são divididas em três grupos: 1. não chocou amostras de referência (definido como 100% de refolded firefly luciferase), 2. células sem chaperone molecular e calor chocado; 3. células com chaperone molecular e calor chocado. </li></ol> 6. Resultados representante Refolding proteína está diretamente relacionada ao tempo de recuperação, portanto, para testar se o sistema está funcionando pro<html> Perly, um ensaio deve ser realizada coleta de células em diferentes momentos. A correlação direta tempo / percentual de refolding indica que o experimento foi devidamente realizado e, portanto, representa um fator limitante. No entanto, deve ser sempre considerado que refolding após choque térmico é um evento saturando e, portanto, uma análise adequada do tempo é claro deve ser realizada antes de iniciar qualquer experimento. <img src="/files/ftp_upload/3540/3540fig1.jpg" alt="Figura 1"/> Figura 1. Visão geral do experimento. Ensaio in vivo de refolding exige 3 a 4 dias para ser concluída. No dia 1 células são semeadas e transfectadas. As células são parceladas dia seguinte em um formato menor e no dia 3 de calor são chocados. Neste ponto é possível realizar o ensaio de luciferase logo após a colheita as células ou para salvar o pellet celular a -80 ° C e realizar o teste em outro momento _content &quot;&gt; <img src="/files/ftp_upload/3540/3540fig2.jpg" alt="Figura 2"/> Figura 2. Divisão celular. No dia 2 células dividem-se em uma placa de seis poços. Cada transfecção será diluído em um poço, a fim de ter todos os transfecção no mesmo prato. Cada placa irá corresponder a um tempo de recuperação especial além de uma placa de referência não chocado. <img src="/files/ftp_upload/3540/3540fig3.jpg" alt="Figura 3"/> Figura 3. Bom resultado Representante. A. Porcentagem de refolding medido diretamente pela atividade firefly luciferase. Cada gráfico de barras representa a porcentagem de refolding na ausência (barras pretas) e na presença (barras vermelhas) de uma chaperone molecular em diferentes momentos de recuperação. B. Correlação entre tempo e redobrando na ausência (linha preta) e na presença (linha vermelha) de acompanhante. Os valores de r ao quadrado indicam boa correlação. Em Fig.3A é mostrado um resultado representativo onde as células foram coletadas 15, 30, 45, 60 e 120 após choque térmico na ausência (barras pretas) e na presença (barras vermelhas) de chaperone molecular. Percentual de refolded aumenta luciferase com o tempo de recuperação prolongado e com a transfecção do chaperone molecular. A correlação entre refolding e tempo para o controle (Fig. 3B, linha preta) e do chaperone molecular células transfectadas (Fig. 3B, linha vermelha) é mostrado. <img src="/files/ftp_upload/3540/3540fig4.jpg" alt="Figura 4"/> Figura 4. Representante mau resultado. A. Porcentagem de refolding medido diretamente pela atividade luciferase. Cada gráfico de barras representa a porcentagem de refolding na ausência (barras pretas) e na presença (barras vermelhas) de uma chaperone molecular em diferentes momentos de recuperação. B. Correlação entre tempo e redobrando na ausência e na presença (vermelho line) de acompanhante. Os valores de r ao quadrado indicam correlação ruim. Figura 4A. Mostra um resultado representativo de uma experiência não adequadamente realizada. Tanto o controle (barras pretas) eo chaperone molecular transfectadas células (barras vermelhas) não mostraram um aumento na proteína refolding com o tempo. Além disso, transfecção do chaperone molecular não resultou em um aumento de renaturação. Esta baixa correlação é confirmada na fig. 4B mostrando um valor de r quadrados de 0,6619 e 0,1882, respectivamente, para o controle (linha preta) e do chaperone molecular transfectadas células (linha vermelha).

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	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

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Neste trabalho um protocolo para medir a atividade refolding intracelular de chaperones moleculares é apresentado. O ensaio completo pode ser realizado em 3 a 4 dias como mostra a visão geral na fig. 1. A robustez ea linearidade do sinal de luz produzida pelo vaga-lume ea luciferase renilla representam uma base sólida para a reprodutibilidade do protocolo. A etapa crítica do ensaio é a escolha de um reagente de transfecção eficiente para g...

Para esta experiência &quot;Luz Vector &#39;a Luminômetro forma Perkin Elmer Contador Luminescência &#39;1420 foi usado Programas para o Luminômetro: Renilla: Bomba 1, 100 mL de injeção Tampão de reação: Pump1, 70 mL de injeção Luciferina: Pump2, a injeção de 30μl Para os buffers preparar estas soluções estoque em água destilada duas vezes: <ul style="list-style:none"><li> - 1M MgSO 4: 246,48 g MgSO 4 * 7H 2 O em 1 litro </li><li> - 0,5 M EGTA: 190,18 g EGTA em 1 litro </li><li> - 1M KH 2 PO 4: 136,09 g KH 2 PO 4 em 1 litro </li><li> - 1M K2 HPO 4: 228,23 g K 2 HPO 4 * 3H 2 O em 1 litro </li><li> - NaCl 5M: 292,2 g de NaCl em 1 litro </li><li> - 0,5 M EDTA: 186,12 g EDTA * 2H 2 O em 1 litro &nbsp;</li></ul> GLI-GLI-buffer: <ul style="list-style:none"><li> - 25mM Glycylglycin </li><li> - 15mm MgSO 4 </li><li> - 4 mM EGTA </li></ul> Para 1 litro use 3,3 g de Glycylglycin, 15ml de uma solução de MgSO4 1M e 8ml de uma solução 0,5 M EGTA. Resolver em água, ajustar o pH para 7,8 e trazer para um volume total de 1 litro. Tampão de reação para Renilla / celenterazina buffer: <ul style="list-style:none"><li> - &amp; Nbsp; 13,4 mM KH 2 PO 4 </li><li> - 86,6 mM KH 2 PO 4 </li><li> - 0,5 M NaCl </li><li> - 1mM EDTA </li></ul> Para 1 litro ml usar 13,4 de um 1M KH 2 PO 4 solução, 86,6 ml de uma 1M K 2 HPO 4 solução, 100ml de uma solução de NaCl 5M e 2ml de uma solução de EDTA 0,5 M. <table border="1"><tr><td></td><td> Companhia </td><td> Catálogo / Produto Número: </td></tr><tr><td> DMEM (Dulbecco Modificado Eagle Medium 1X) </td><td> GIBCO </td><td> 41966029 </td></tr><tr><td> Ouro de soro fetal bovino </td><td> PAA </td><td> A15-151 </td></tr><tr><td> X-treme Gene 9 Reagente Transfection DNA </td><td> Roche </td><td&gt; 06365809001 </td></tr><tr><td> PBS Phosphate Buffered Saline Dulbecco 1X </td><td> GIBCO </td><td> 14190-094 </td></tr><tr><td> MOPS 3 - (N-Morfolino) Propanesulfonic ácido </td><td> SIGMA-ALDRICH </td><td> M1254 </td></tr><tr><td> Cicloheximida </td><td> SIGMA-ALDRICH </td><td> C7698 </td></tr><tr><td> Tampão de Lise passiva 5X </td><td> Promega </td><td> E194A </td></tr><tr><td> Glycylglycin EGTA MgSO 4 (MgSO 4 * 7H 2 O) </td><td> SIGMA-ALDRICH Roth Roth </td><td> G7278 3054,2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4 (K 2 HPO 4 * 3H 2 O) NaCl EDTA </td><td> Roth Roth Roth Roth </td><td> 3904,1 6878,2 3957,1 8043,1 </td></tr><tr><td> Luciferina Firefly </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> Celenterazina </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT (Ditiotreitol) </td><td> Roth </td><td> 6908,2 </td></tr><tr><td> ATP (adenosina trifosfato) </td><td> Roche </td><td> 10519987001 </td></tr></table>

intracellular refolding assay

Este protocolo descreve um método para medir a atividade enzimática de chaperones moleculares em um sistema baseado em células e os possíveis efeitos de compostos com atividade inibitória / estimulante. Chaperones moleculares são proteínas envolvidas na regulação do enovelamento de proteínas 1 e têm um papel crucial na promoção da sobrevivência celular após insultos estresse, como choque térmico 2, a fome de nutrientes e exposição a produtos químicos / venenos 3. Por esta razão chaperones são encontrados para ser envolvido em eventos como o desenvolvimento do tumor, chemioresistance das células cancerosas 4, bem como neurodegeneração 5. Desenho de pequenas moléculas capazes de inibir ou estimular a atividade destas enzimas é, portanto, uma das estratégias mais estudadas para terapia de câncer e doenças neurodegenerativas 7 9. O ensaio aqui descrito oferece a possibilidade de medir a atividade refolding de um chaperone molecular particular e para estudar o efeito da compounds sobre a sua actividade. Neste método o gene da chaperone molecular investigado é transfectadas com uma codificação de vetor de expressão para o gene luciferase vaga-lumes. Já foi descrito que a luciferase firefly desnaturado podem ser reabertos por chaperones moleculares 10,11. Como normalizar o controle de transfecção, uma codificação de vetor para o gene luciferase é renilla transfectadas. Todos os transfections descritos neste protocolo são realizadas com X-treme Gene 11 (Roche) em células HEK-293. Na primeira etapa, a síntese protéica é inibida pelo tratamento das células com cicloheximida. Posteriormente proteína desdobramento é induzida por choque térmico a 45 ° C por 30 minutos. Após a recuperação, a 37 ° C, as proteínas são re-dobrada em sua conformação ativa e da atividade da luciferase firefly é usado como ler-out: quanto mais luz será produzido, o mais proteína terá re-ganhou a conformação original. Não-calor células chocado são definidos como referência (100% de refoldedluciferase).

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

Neste protocolo um método para medir refolding proteína intracelular após choque térmico é descrito. Este método pode ser usado para estudar foldases como chaperonas moleculares e seus co-fatores ou compostos capazes de influenciar a sua actividade. Firefly luciferase atividade é utilizado como repórter para medir a atividade refolding acompanhante.

Ensaio redobrando intracelular

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

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JoVE Journal

Biology

14.0K visualizações.  Karlsruhe Institute of Technology. Neste protocolo um método para medir refolding proteína intracelular após choque térmico é descrito. Este método pode ser usado para estudar foldases como chaperonas moleculares e seus co-fatores ou compostos capazes de influenciar a sua actividade. Firefly luciferase atividade é utilizado como repórter para medir a atividade refolding acompanhante.

Vídeo: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Medição Perto membrana plasmática e global Dynamics cálcio intracelular em astrócitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biologia

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Imaging o tráfego intracelular de APP com GFP fotoactivável

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificação e redobramento de amilóide fibrilas de (seu)<sub&gt; 6</sub&gt; tagged proteína recombinante Shadoo Expresso como Corpos de Inclusão em<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Identificação de sinalização intracelular Eventos induzida em células viáveis ​​pela interação com as células vizinhas Submetidos a apoptose celular Morte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

PH intracelular de quantificação de óptica em Drosophila melanogaster túbulo de Malpighi epitélios com um indicador de pH fluorescente geneticamente codificado

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Lente livre de microscopia para a dinâmica e a análise quantitativa de cultura de células aderentes

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...