Somos gratos ao Departamento de Fisiologia e Biologia Molecular, Universidade da Califórnia Davis por nos fornecer instalações e um excelente ambiente para os estudos de canais iônicos.

1. Preparação de repouso Linfócitos T Humanos <ol><li> Usando RosetteSep Humanos Cocktail de Enriquecimento de células T e Medium Density RosetteSep, purificar as células T a partir de amostras de sangue humano de acordo com as instruções do fabricante. A população de células resultante deve conter 95% CD3 + células T em repouso. Purificamos linfócitos T humanos a partir de amostras coletadas do sangue periférico de voluntários saudáveis, em conformidade com o protocolo aprovado pelo Internal UC Davis Board Review. </li><li> Após o isolamento, descansando ressuspender as células T com uma densidade de 0,5 x 10 6 células / mL em meio de cultura de células contendo RPMI-1640 meio com glutamina e HEPES, suplementado com 10% de soro fetal bovino, 2% Glutamax-I, 1% RPMI 1640 vitamina solução, 1% solução de RPMI 1640 aminoácidos, piruvato de sódio a 1%, e 0,03% β-mercaptoetanol. </li><li> Suspensão lugar célula em frascos de cultura de células e manter a 37 ° C em uma incubadora umidificada com 5% de CO 2 para até 24 h antes do experimento. </li></ol> 2. Preparação da Câmara de gravação <ol><li> A seguir são necessários para preparar as câmaras de gravação: a) de silício O-rings, b) redondo, 25 mm lamínulas limpos com álcool 70% e destilada H 2 O, c) mista Sylgard184, preparados de acordo com as instruções do fabricante, e d) dois 60 x 15 mm placas de Petri. </li><li> Coloque ~ 0,5 mL de Sylgard184 misturado em uma placa de Petri 60x15 mm. </li><li> Mergulhe a parte inferior do O-ring para o forceps Sylgard184 usando. </li><li> Retire o excesso de Sylgard184 da borda do anel, colocando-o O-ring em uma superfície limpa, como uma outra placa de Petri. </li><li> Coloque o anel sobre a lamínula e pressione-o para baixo. </li><li> Curar a Sylgard184 pelo aquecimento das câmaras a 85 ° C por 40-60 min ou até que o Sylgard184 é polimerizado. </li><li> Armazenar as câmaras de gravação em um recipiente de pó. </li></ol> 3. Preparação de Pipetas patch <ol><li> No dia do experimento, puxe pipetas com um diâmetro da ponta ~ 2 mícrons usando capilares de vidro e um puxador de pipeta. Usamos tubos de borosilicato com filamento (OD: 1,5 mm e ID: 1.10 mm). Pipetas loja em um local livre de poeira. </li><li> Brasão da pipeta pontas com Sylgard184 ou revestimento Semiconductor Hipec R6101 de proteção, de acordo com um procedimento padrão 9. </li><li> Suavemente-fogo polonês as pipetas antes do experimento. </li></ol> 4. Remendo Preparação Setup Grampo <ol><li> Configurar e salvar as macros para a formação gigaseal no software de controle de seu amplificador de patch-clamp. Nós usamos um amplificador EPC-10 suportados pelo Windows XP e software de pulso para a aquisição de dados. Vamos definir o &quot;set-up&quot;, &quot;na célula&quot;, e &quot;todo-cell&quot; macros de acordo com o manual de EPC-10 e usá-las para todos os experimentos. </li><li> Preparar soluções de banho e soluções pipeta listadas na Tabela 1 antes do experimento e armazená-los a 4 ° C por até 1 semana. </li></ol><table border="1"><tbody><tr><td> Produtos químicos </td><td colspan="3"> Soluções de banho (mM) </td><td> Pipeta Solutions (mM) </td></tr><tr><td></td><td> # 1 </td><td> # 2 </td><td> # 3 </td><td></td></tr></tbody><tbody><tr><td> CH 3 SO 3 Na </td><td> 130 </td><td> 110 </td><td> 125 </td><td> - </td></tr><tr><td> NaCl </td><td> 2 </td><td> 4 </td><td> 5 </td><td> - </td></tr><tr><td> Ca (OH) 2 </td><td> - </td><td> 20 </td><td> - </td><td> - </td></tr><tr><td> MgCl 2 </td><td> 3 </td><td> 1 </td><td> - </td><td> 5 </td></tr><tr><td> MgSO 4 </td><td> - </td><td> - </td><td> - </td><td> 2 </td></tr><tr><td> HEPES </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 15 </td></tr><tr><td> HEDTA </td><td> - </td><td></td><td> 10 </td><td> - </td></tr><tr><td> EDTA </td><td> - </td><td> - </td><td> 1 </td><td> - </td></tr><tr><td> Cs-aspartate </td><td> - </td><td> - </td><td> - </td><td> 125 </td></tr><tr><td> BAPTA </td><td> - </td><td> - </td><td> - </td><td> 12 </td></tr><tr><td> Glicose </td><td> 10 </td><td> 10 </td><td> 10 </td><td> - </td></tr></tbody></table> Tabela 1. Soluções para toda célula CARC gravações atuais. Todos os produtos químicos foram adquiridos da Sigma-Aldrich, St. Louis, MO. <ol start="3"><li> Determinar potenciais de junção líquida (LJ) entre a solução de pipeta patch e cada solução de banho de 10. Salvar o valor LJ no local apropriado utilizar o seu software de aquisição. Por exemplo, no programa Pulso inserir o valor LJ em &quot;LJ&quot; no controle &quot;amplificador&quot; janela. </li><li> Configurar e salvar os protocolos de estimulação, como mostrado na Figura 1, no local apropriado do seu software de aquisição antes dos experimentos. No programa Pulse, vamos definir o protocolo de aquisição na &quot;Geração Pulse&quot; janela. <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="Figura 1" /> Figura 1. . Protocolo de estimulação Rampa de tensão de -120 a +100 mV de 50 ms de duração é aplicado a cada 0,2-2 s (5-0,5 Hz). Potencial de exploração (V h) entre rampas é mantida em 30 mV. </li></ol> 5. As células de montagem no Palco Microscópio <ol><li> Revestimento das câmaras de gravação com poli-L-lisina para ~ 20 minutos e lave com H 2 O e depois com solução salina balanceada de Hank (HBSS) antes do plaqueamento das células. </li><li> ML placa de 500 de suspensão de células contendo 0,2-0,5 x10 6 células na câmara e incubar por 20 min a 37 ° C. </li><li> Segura a câmara de gravação com as células anexado em um suporte de câmara. Usamos um suporte de câmara custom-made, onde a base de suporte na parte inferior da lamela, enquanto a parte de cima delicadamente pressiona contra a parte superior da lamínula. </li><li> Coloque o suporte da câmara no palco do microscópio invertido. </li><li> Foco sobre as células em luz transmitida. Nós usamos uma objetiva de imersão 40X de petróleo. </li><li> Coloque o eletrodo de referência Ag / AgCl para o banho. </li><li> Alinhar o multibarrel, sistema de perfusão gravidade-driven. A ponta do tubo de entrada deve ser colocado em um ângulo de 45 ° até o fim lamela para o campo de visão. </li><li> Posicione o tubo de sucção em frente à ponta do tubo de entrada. </li><li> Perfundir a câmara com tampão fosfato (PBS) ou HBSS para lavar os meios de cultura celular e células frouxamente ligados. </li></ol> 6. CRAC Canal Gravação Correntes <ol><li> Encontrar uma célula que está firmemente ligado à lamela. Nós geralmente seleciona uma média de tamanho de célula, redonda com uma superfície lisa exterior. </li><li> Perfundir da câmara gravando com Ca 2 + livre, 3 mM Mg 2 + solução de banho contendo # 1 (Tabela 1) contendo 0,5-1 tapsigargina M para 80-10 min para bloquear a bomba SERCA. Realizamos este passo para empobrecem a loja antes da formação gigaseal. </li><li> Usando um microloader Eppendorf e um P-20 pipeta deslocamento, encher a pipeta patch com a solução da pipeta (Tabela 1) e inserir a pipeta patch para o titular pipeta na headstage amplificador. O Ca 2 + quelante BAPTA está incluído na solução de pipeta para passivamente empobrecem a loja e para reduzir o Ca 2 + dependentes de inativação do canal CRAC. </li><li> Aplique uma pequena quantidade de pressão positiva (~ 3 cm de H 2 O) no interior da pipeta de patch antes de entrar no banho. Nós usamos um dispositivo feito sob medida de pressão de sucção conectado ao ar pressurizado e linhas de vácuo para criar uma pressão positiva ou negativa dentro de uma pipeta patch. </li><li> Inferior da pipeta patch para o banho sob controle visual através do microscópio. No &quot;Osciloscópio&quot; janela, você deve ver uma corrente que flui através da pipeta. A mudança de patamar-como na amplitude da corrente deve ser induzido por um pulso de pequena amplitude da tensão (teste de pulso), aplicadas pela macro predefinida. Neste momento, você deve ser capaz de determinar o valor da resistência da pipeta. A resistência de nosso pipetas é tipicamente 5-6 mohms. </li><li> Corrigir o &quot;offset&quot; potencial gerado entre a pipeta eo eletrodo de referência. </li><li> Sob controle visual através do microscópio, traga a ponta da pipeta mais próximo da célula até tocar a membrana celular. </li><li> Aplicar pressão negativa (20-40 polegadas de H 2 O) no interior da pipeta patch. </li><li> Monitorar a formação gigaseal no &quot;osciloscópio&quot; janela. Você vai ver a amplitude da corrente, induzido pelo pulso de teste, diminui e aumenta o valor da resistência pipeta&gt; 5 GÊ. Formas normalmente gigaseal dentro de um fesegundo w, mas pode demorar 1-2 minutos para conseguir um gigaseal estável. </li><li> Compensar a capacitância pipeta (&quot;C-Fast&quot;). </li><li> Ruptura da membrana correção através da aplicação de pressão negativa adicional, usando uma seringa de 20 cc. </li><li> Definir o potencial de exploração para 30 mV. Um potencial de detenção positivo ajuda a prevenir cálcio-dependente inativação do canal CRAC. </li><li> Compensar a capacitância da membrana da célula (&quot;C-slow&quot;); salvar ou gravar o valor de C-lenta. </li><li> Verificar o valor da resistência de acesso &quot;R s&quot;. Em nossos experimentos, o s R é tipicamente 70-20 mohms. </li><li> Começar a aplicar uma série de rampas de tensão com uma freqüência de 0,5 Hz (Figura 1) em Ca 2 + livre mM 3 Mg 2 + solução de banho contendo o 1 º. Nesta solução, atual CRAC é pequeno em comparação com o &quot;vazamento&quot; de corrente devido à pouca permeabilidade dos canais CRAC para Mg 2 +. Salvar e usar os traços atuais gravadas em solução de banho # 1 em análises futuras como &quot;vazamento&quot; das correntes. A amplitude absoluta do &quot;vazamento&quot; corrente a -100 mV deve ser menor ou igual a 5 pA. Se a célula é &quot;furado&quot;, interromper a experiência e começar de novo com uma pipeta de novo adesivo e uma nova célula. </li><li> Para gravar Ca 2 + através de canais atuais CRAC (I Ca-CRAC), substituir Ca 2 + livre 3 mM Mg 2 + solução de banho contendo # 1 com 20 mM Ca 2 + contendo solução de banho # 2 (Tabela 1), alternando as válvulas de perfusão. Isto permite Ca 2 + a fluir através de canais de CRAC e produzir uma corrente dentro. No &quot;Osciloscópio&quot; janela, você verá o desenvolvimento de retificar interiormente eu Ca-CRAC (Figura 2). A amplitude da corrente com tensões negativas continuará a aumentar para cerca de 1 min, devido ao Ca 2 + dependentes de potenciação da corrente CRAC. </li><li> Aumentar a freqüência de estimulação com rampas de tensão para 5 Hz, que é necessário para gravar o Na + transitória atuais através de canais CRAC. </li><li> Substituir os 20 mM Ca 2 + contendo solução de banho # 2 com Na +-ção contendo solução livre de bivalentes # 3 (Tabela 1), alternando as válvulas de perfusão. No &quot;Osciloscópio&quot; janela, você verá um desenvolvimento transitório de maior amplitude dentro retificar Na + atual através dos canais CRAC (I Na-CRAC; Figura 2). </li><li> Pode-se aplicar 20 mM Ca 2 + contendo solução de banho # 2 contendo 1-5 mM La 3 +, no final do experimento para gravar o &quot;vazamento&quot; atual. No entanto, não encontramos essa abordagem útil porque o &quot;vazamento&quot; mudanças em curso ao longo do tempo e pode se tornar maior ou menor no final do experimento. Nós preferimos tomar a atual registrado no Ca 2 + livre mM 3 Mg 2 + contendo solução de banho de # 1 no início do experimento como uma &quot;fuga&quot; atual. </li><li> Guardar os vestígios gravados atual para posterior análise. </li></ol> 7. Análise de Dados <ol><li> Recuperar os registros salvos atual em software de análise. Usamos o programa de pulso para análise. </li><li> Medir a amplitude atual no início e no final das rampas de tensão. Normalmente, medir a amplitude atual em -100 mV e em 100 mV usando pares de cursores no &quot;Osciloscópio&quot; janela do programa Pulse. </li><li> Exportar os valores de amplitudes atual em um programa gráfico para posterior análise e apresentação gráfica. Usamos Graphing Origem Científica e Software Análise, versão 7. </li></ol> 8. Resultados representante <img src="/files/ftp_upload/2346/2346fig2.jpg" alt="Figura 2" /> Figura 2. Correntes CRAC em uma célula T humano descansando. (A), o curso Tempo de correntes CRAC registrados em todo o celular de configuração de voltagem clamp-a -100 mV (círculos cheios) e 100 mV (círculos abertos). Antes da formação gigaseal, a célula foi pré-incubado por ~ 10 min em Ca 2 + livre mM 3 Mg 2 + contendo solução de banho # 1 contendo 0,5 mM tapsigargina. Após invasão, soluções de banho foram seqüencialmente aplicados da seguinte forma: Ca 2 + livre 3 mM Mg 2 + contendo solução de banho # 1 (0 Ca + Mg 3), seguido por 20 mM Ca 2 + solução de banho contendo # 2 (20 Ca), seguido por divalente-cação solução livre de banho # 3 (DVF), seguido pela solução de banho # 2 (20 Ca). A célula foi estimulado com uma série de rampas de tensão, como mostrado na Figura 1. A freqüência de rampas foi de 5 Hz em solução de banho # 3 (DVF) e 0,5 Hz em todas as outras soluções. Observe o lento desenvolvimento de I Ca-CRAC após a aplicação de 20 mM Ca 2 + contendo solução de banho # 2 eo rápido desenvolvimento transitória da I Na-CRAC na DVF solução de banho # 3. (B), Representante traços corrente registou durante rampas de tensão em Ca 2 + livre 3 mM Mg 2 + solução de banho contendo # 1 (&quot;Vazamento&quot;), 20 mM Ca 2 + contendo solução de banho # 2 (20 Ca), e uma solução de banho # 3 (DVF). (C, D), atual tensão relações de I Ca-CRAC (C) e eu Na-CRAC (D) obtida subtraindo-se &quot;vazar&quot; atual das correntes registradas durante tensão rampas em 20 mM Ca 2 + contendo banho solução # 2 (20 Ca), e uma solução de banho # 3 (DVF) mostrado no painel (B).

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+channel+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

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A investigação eletrofisiológicos de correntes CRAC em repouso células T humanas é uma tarefa desafiadora, porque a amplitude CRAC de corrente endógena dessas células é pequeno devido ao tamanho de pequenas células (o diâmetro de células T humanas em repouso está na faixa de 5-8 mm). Aqui, apresentamos um procedimento passo-a-passo para registro confiável correntes CRAC em repouso linfócitos T humanas isoladas a partir de células mononucleares do sangue periférico. Esta tecnologia permite-nos investigar ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
<tr>
<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
<tr>
<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
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whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

Em linfócitos T, a depleção de Ca 2 + intracelular do Ca 2 + loja leva a ativação de plasmalemmal Ca 2 + canais, chamados de cálcio Release-Activated cálcio (CRAC) canais. CRAC canais desempenham um papel importante na regulação da proliferação de células T e expressão gênica. Função de canal de anormal CRAC nas células T tem sido associada a doenças de imunodeficiência combinada severa e auto-imunes 1, 2. Estudar a função de canal CRAC em células humanas T pode descobrir novos mecanismos moleculares que regulam as respostas imunes normais e desvendar as causas de doenças relacionadas com o humano. Gravações eletrofisiológicas das correntes de membrana uma avaliação mais precisa das propriedades do canal funcional e sua regulamentação. Avaliação eletrofisiológica das correntes CRAC canal em células Jurkat T, um ser humano da leucemia de células T de linha, foi realizado mais de 20 anos atrás 3, no entanto, as medições atuais CRAC em condições normais de células T humanas permanece uma tarefa desafiadora. As dificuldades na gravação correntes canal CRAC em células T normais são agravados pelo fato de que os linfócitos T derivados do sangue são muito menores em tamanho do que as células T Jurkat e, portanto, o endógeno correntes de célula inteira CRAC são muito baixos em amplitude. Aqui, damos um procedimento passo-a-passo que usam rotineiramente para gravar o Ca 2 + ou Na + correntes através de canais CRAC em repouso células T humanas isoladas do sangue periférico de voluntários saudáveis. O método descrito aqui foi adotado a partir dos procedimentos utilizados para a gravação das correntes CRAC em células Jurkat T e ativação de células T humanas 4-8.

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Nós fornecemos um protocolo passo a passo para a gravação de célula inteira patch clamp de Cálcio O cálcio Release-Ativado (CRAC) correntes em sangue periférico mononuclear de células derivadas de linfócitos T humanos.

Todo-Cell Gravação de Cálcio O cálcio Release-Ativado (CRAC) Correntes em linfócitos humanos T

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

whole-cell-recording-calcium-release-activated-calcium-crac-currents

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

16.0K visualizações.  University of California, Davis. Nós fornecemos um protocolo passo a passo para a gravação de célula inteira patch clamp de Cálcio O cálcio Release-Ativado (CRAC) correntes em sangue periférico mononuclear de células derivadas de linfócitos T humanos.

Vídeo: Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Carregando Drosophila terminais nervosos com indicadores de cálcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Biologia

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

Vivo em 2-Photon Imagens de cálcio na camada 2 / 3 dos Ratos

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

De célula inteira Recordings of Light Evocados Excitatórios Correntes Synaptic na Slice Retinal

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

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, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Fluxos de medição de cálcio rápido no Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Gravação em tempo real na Vivo de Arabidopsis sinais de cálcio durante a alimentação de insetos usando um biossensor fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...