Este estudo foi suportado por concessões do subsídio do cientista proeminente de Shandong (JQ201421) e bolsa da NSFC (81371226).

1. isolamento de mitocôndrias
Nota: Todas as soluções e equipamentos devem ser pré-resfriado a 0 - 4 ° C e mantidos no gelo.
<ol><li>Semente cerca de 3 x 106 SH-SY5Y células por placa de cultura de células de 10 cm. Células de cultura em alta-glicose Dulbecco modificada do meio de águia com 10% de soro bovino fetal, penicilina (100 U/mL)-estreptomicina (100 µ g/mL). Manter a 37 ° C numa incubadora contendo 5% CO2 durante a noite. Nota: pelo menos 1-2 x 107 SH-SY5Y de células são necessárias para cada ensaio de isolamento.</li>
	<li>Retire o meio e lave as células com cerca de 1 mL de PBS frio de gelo em um prato de cultura de célula de 10cm 3 vezes.</li>
	<li>Adicionar novo 1 mL de PBS frio gelo em placa de cultura de células de 10 cm e raspar as células aderentes em um novo tubo de 1,5 mL. Centrifugar 800 x g por 10 min a 4 ° C.</li>
	<li>Durante a centrifugação, prepare-se 5 mL de tampão de isolamento mitocondrial (250 mM sacarose, 10 mM HEPES (ácido de 4-(2-hydroxyethyl)-1-piperazina etanesulfónico), 10 mM KCl, 1,5 mM de MgCl2, 1 mM EDTA) suplementado com 50 μL de estoque de inibidores de protease solução (concentração de x 100; 1 comprimido de EDTA livre dissolvido em 500 µ l do ddH2O).</li>
	<li>Remover o sobrenadante e ressuspender o precipitado com 1 mL de tampão de isolamento mitocondrial. Incube o tubo de 1,5 mL no gelo por 30 min.</li>
	<li>Lise as células suspensas com um homogeneizador de vidro (traços de 15-25) acima e para baixo manualmente no gelo. Nota: Certifique-se de que não há nenhuma bolha quando as células são homogeneizadas. Contar as células intactas e núcleos exposto pode por microscópio visualmente e confirmar que > 90% célula ruptura já ocorreu.</li>
	<li>Transferi o homogeneizado em um tubo de 1,5 mL e centrifugar a 1.000 x g por 5 min a 4 ° C para remover os detritos (núcleos e células intactas remanescentes).</li>
	<li>Transferir o sobrenadante para um novo tubo de 1,5 mL e centrifugar a 1.000 x g por 5 min 4 ° C.</li>
	<li>Transferir o sobrenadante para um novo tubo e centrifugar 14.000 x g por 15 min 4 ° C.</li>
	<li>Remover o sobrenadante e ressuspender o precipitado que contém mitocôndrias com 1 mL de tampão de isolamento mitocondrial.</li>
	<li>Centrifugue a solução por 15 min a 14.000 x g 4 ° C para precipitar as mitocôndrias.</li>
	<li>A pelota de mitocôndrias resultante deve ser mantida no gelo e resuspended em 50-100 μL KCl media (125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 20mm HEPES, glutamato de 5mm, malato de 5mm e 2 a rotenona μM, pH 7,4 de acordo com o volume de sedimento antes de qualquer rabo) Ay.</li>
	<li>Use 5 μL de solução mitocondrial para medir a concentração de proteína mitocondrial por padrão ensaio BCA, OD562 de medição usando um leitor de placa13.</li>
</ol>2. determinação da mitocondrial Ca2 + capacidade de retenção
<ol><li>Preparar a mídia de KCl e adicionar o Ca2 +-ligação corante fluorescente verde a uma concentração final de 0,5 μM antes do experimento.</li>
	<li>Transferir 1 mL de mitocôndrias isoladas (0,4 mg/mL) em mídia de KCl contendo 0,5 μM Ca2 +-ligação corante fluorescente verde em cada placa de 6 bem.</li>
	<li>Incubar as mitocôndrias e o Ca2 +-ligação corante fluorescente verde na placa 6-poços em temperatura ambiente protegida da luz ambiente para 1 min. Adicione 4 alíquotas μL de uma solução de CaCl2 (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2 de 20 mM 20 mM HEPES, glutamato de 5mm, malato de 5 mM, pH 7,4) para cada placa de 6 bem usando o automático dispensa configuração apresentar 200 nmol Ca2 +/ mg proteína mitocondrial.</li>
	<li>Usar um espectrômetro de fluorescência para monitorar as alterações de fluorescência cada 3 s por 2 min, com um comprimento de onda de excitação de 506 nm e um comprimento de onda de emissão de 531 nm. A placa é equipada com tremendo a 600 rpm por 3 s entre leituras controlada por software (Figura 1).</li>
	<li>Adicionar a alíquota adicional 4 μL da solução de CaCl2 de 20 mM a cada poço e monitorar as mudanças de fluorescência cada 3 s por 2 min. Repita este passo até que a fluorescência aumenta com o tempo. Nota: As mitocôndrias são desafiadas com o adicionado Ca2 + indicado por fluorescência o aumento de gravação. Quando o mPTP abre, a fluorescência aumenta com o tempo.</li>
	<li>Interprete a quantidade total de Ca2 + adicionado até o mPTP abre como a capacidade de Ca2 + de retenção.</li>
</ol>3. determinação de Ca 2 +-induzido por inchaço mitocondrial
<ol><li>Prepare a mídia de KCl antes do experimento.<ol>
<li>Transferi 1 mL de mitocôndrias isoladas (0,4 mg/mL) em mídia de KCl para cada placa de 6 bem.</li>
	</ol>
</li>
	<li>Adicione 10 μL de solução aquosa de CaCl2 (500 nmol/mg de proteína mitocondrial) de 20 mM em proteína mitocondrial para acionar o inchaço mitocondrial.</li>
	<li>Gravar a diminuição da absorbância em 540 nm cada 3 s em um leitor de microplacas controlado por software para 10 min (Figura 2). As alterações de fluorescência são causadas por um afluxo de solutos através da membrana mitocondrial interna. Nota: A diminuição da absorbância em 540 nm corresponde à mitocôndria inchadas. Se o leitor não observar uma diminuição na absorção, um longo intervalo de leitura ou tempo de leitura pode ser adotado.</li>
</ol>

<ol>
	<li>Rossi, C. S., Lehninger, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+respiratory+stimulation,+accumulation+of+Ca+++and+phosphate,+and+oxidative+phosphorylation+in+rat+liver+mitochondria.">Stoichiometry of respiratory stimulation, accumulation of Ca++ and phosphate, and oxidative phosphorylation in rat liver mitochondria.</a> J Biol Chem. 239 (11), 3971-3980 (1964).</li><li>Gunter, T. E., Buntinas, L., Sparagna, G., Eliseev, R., Gunter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+transport:+mechanisms+and+functions.">Mitochondrial calcium transport: mechanisms and functions.</a> Cell Calcium. 28 (5-6), 285-296 (2000).</li><li>Crofts, A. R., Chappell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Ion+Accumulation+and+Volume+Changes+of+Isolated+Liver+Mitochondria.+Reversal+of+Calcium+Ion-Induced+Swelling.">Calcium Ion Accumulation and Volume Changes of Isolated Liver Mitochondria. Reversal of Calcium Ion-Induced Swelling.</a> Biochem J. 95, 387-392 (1965).</li><li>Wojda, U., Salinska, E., Kuznicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+ions+in+neuronal+degeneration.">Calcium ions in neuronal degeneration.</a> IUBMB Life. 60 (9), 575-590 (2008).</li><li>Celsi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+calcium+and+cell+death:+a+deadly+triad+in+neurodegeneration.">Mitochondria, calcium and cell death: a deadly triad in neurodegeneration.</a> Biochim Biophys Acta. 1787 (5), 335-344 (2009).</li><li>Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., Fiskum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+potentiates+the+maximal+calcium+uptake+capacity+of+neural+cell+mitochondria.">Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.</a> Proc Natl Acad Sci U S A. 93 (18), 9893-9898 (1996).</li><li>Rajdev, S., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+green-5N,+a+novel+fluorescent+probe+for+monitoring+high+intracellular+free+Ca2++concentrations+associated+with+glutamate+excitotoxicity+in+cultured+rat+brain+neurons.">Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons.</a> Neurosci Lett. 162 (1-2), 149-152 (1993).</li><li>Stout, A. K., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+calcium+indicators+underestimate+increases+in+intracellular+calcium+concentrations+associated+with+excitotoxic+glutamate+stimulations.">High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations.</a> Neuroscience. 89 (1), 91-100 (1999).</li><li>Bambrick, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A+increases+mitochondrial+calcium+uptake+capacity+in+cortical+astrocytes+but+not+cerebellar+granule+neurons.">Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.</a> J Bioenerg Biomembr. 38 (1), 43-47 (2006).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-permeable+peptide+antioxidants+targeted+to+inner+mitochondrial+membrane+inhibit+mitochondrial+swelling,+oxidative+cell+death,+and+reperfusion+injury.">Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury.</a> J Biol Chem. 279 (33), 34682-34690 (2004).</li><li>Muriel, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+free+calcium+levels+(Rhod-2+fluorescence)+and+ultrastructural+alterations+in+neuronally+differentiated+PC12+cells+during+ceramide-dependent+cell+death.">Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.</a> J Comp Neurol. 426 (2), 297-315 (2000).</li><li>Allman, R., Hann, A. C., Phillips, A. P., Martin, K. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Azotobacter+vinelandii+with+correlation+of+Coulter+cell+size,+flow+cytometric+parameters,+and+ultrastructure.">Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.</a> Cytometry. 11 (7), 822-831 (1990).</li><li>Wiechelman, K. J., Braun, R. D., Fitzpatrick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+bicinchoninic+acid+protein+assay:+identification+of+the+groups+responsible+for+color+formation.">Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation.</a> Anal Biochem. 175 (1), 231-237 (1988).</li><li>Petronilli, V., Nicolli, A., Costantini, P., Colonna, R., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+permeability+transition+pore,+a+voltage-dependent+mitochondrial+channel+inhibited+by+cyclosporin+A.">Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.</a> Biochim Biophys Acta. 1187 (2), 255-259 (1994).</li><li>Pastorino, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+consequences+of+the+sustained+or+transient+activation+by+Bax+of+the+mitochondrial+permeability+transition+pore.">Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.</a> J Biol Chem. 274 (44), 31734-31739 (1999).</li><li>Gunter, T. E., Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+possible+functions+of+mitochondrial+Ca(2+)+transport+mechanisms.">Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.</a> Biochim Biophys Acta. 1787 (11), 1291-1308 (2009).</li></ol>

X. sol supervisionou os experimentos. Li Wei realizados os experimentos. Zhang Chen e X. sol escreveu o jornal.

Aqui, descrevemos um protocolo simples e eficaz para o mitocondrial Ca2 + retenção capacidade do ensaio e Ca2 +-disparado o ensaio de inchamento mitocondrial.
Para o isolamento mitocondrial, certifique-se de que todos os materiais e os tubos são no gelo, especialmente quando homogeneizando as células. 0,25% do trypsin-EDTA também pode ser usado para destacar células do prato por 5 min a 37 ° C. Depois de 15-25 cursos, é necessário observar a membrana celular inta...

As mitocôndrias são os principais órgãos celulares para produzir cerca de 95% do ATP usado nas células de mamíferos por fosforilação oxidativa. É relatado que o sequestrado micromolar concentração de Ca2 + pela mitocôndria, a presença de ADP e fosfato inorgânico pode ser usado para fosforilar ADP a síntese de ATP1. Quando a concentração de citosólico Ca2 + vai acima de um limite, a mitocôndria pode absorção Ca2 + rapidamente e efluxo isso lentamente. Assim, as mitocôndrias funcionamento podem influxo o aumento citosólico Ca2 +. Irrelevante para a participação na fosforilação oxidativa, o mitocondrial Ca2 + também participar os sinais de cálcio citosólico e ativar o mecanismo de apoptose mitocondrial induzindo a abertura do mPTP no interno mitocondrial membrana2. Foi amplamente reconhecido que elevação anormal de intracelular Ca2 + pode induzir edema maciça mitocondrial abrindo o mPTP3. Assim, o presente protocolo tem por objetivo avaliar a função mitocondrial através da quantificação da capacidade mitocondrial Ca2 + de retenção e Ca2 +-trigged inchaço mitocondrial.
CA2 + capacidade de retenção é a medição da capacidade das mitocôndrias de cálcio citosólico de captação. As mitocôndrias podem buffer o cálcio citosólico livre e regulam processos celulares de cálcio-dependente pela absorção de cálcio na forma de precipitados inativos. Capacidade prejudicada Ca2 + retenção ocorre em fenômenos de estresse associados com limitação de energia e de4,de doenças neurodegenerativas até5. Mitocôndrias isoladas ou células digitonin-permeabilizado podem ser usadas para identificar a capacidade de Ca2 + de retenção, e a elevada capacidade de acumular Ca2 + por mitocôndrias isoladas com o glutamato additonal e malato não é efectuada pelo procedimento de isolamento mitocondrial6. Uma quantia titulada de digitonin deve ser usada para permeabilize as membranas de plasma de diferentes tipos de células. O sal de hexapotassium de Ca2 +-ligação corante fluorescente verde, uma Ca2 +-impermeant-célula visível luz-excitável indicador sensível, tem sido amplamente utilizado7. CA2 +-ligação verde tintura fluorescente é um indicador de baixa afinidade e usado para mostrar que intracelular livre Ca2 + concentrações continuarem a subir durante a prolongada de estímulos (5 min)8. Este método era menos sensível do que a técnica de filtro radioativo mas foi bastante simplificado. O adicional Ca2 + eleva a fluorescência verde do cálcio e da Ca2 + adopção pelas mitocôndrias retorna a fluorescência de base. Adições sequenciais de Ca2 + foram feitas até as mitocôndrias não conseguiram captação extramitochondrial Ca2 + 9. Um leitor de microplacas de fluorescência pode ser usado para relatar continuamente a fluorescência verde de cálcio.
Após o acúmulo de Ca2 + , mitocôndrias despolarizada, liberado Ca2 + para o meio e começaram a inchar. CA2 +-desencadeada mitocôndrias inchaço é usado para indicar a abertura do mPTP. Microscopia eletrônica e a diminuição da luz absorbância em 540 nm pode ser usado para medir o Ca2 +-desencadeada mitocondrial inchaço10,11. Volume de mitocôndrias pode ser diretamente determinada pelo ângulo frente espalhamento de luz12, onde diminui em absorvância reflete passivo inchaço da matriz mitocondrial.
Aqui, podemos ilustrar a metodologia para examinar a capacidade mitocondrial Ca2 + de retenção e Ca2 +-provocou inchaço mitocondrial em mitocôndrias isoladas de SH-SY5Y de células.

<table>
	<tbody>
		<tr>
			<td>SH-SY5Y cell line</td>
			<td>ATCC (The Global Bioresource Center)</td>
			<td>ATCC Number: CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium green-5N</td>
			<td>Life Technologies</td>
			<td>c-3737</td>
			<td>Ca2+-binding green fluorescent dye</td>
		</tr>
		<tr>
			<td>complete protease inhibitors</td>
			<td>Roche Molecular Biochemicals</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>glass homogenizer</td>
			<td>Kimble Chase</td>
			<td>9885303002</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>RES5063G-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>46940-U</td>
			<td></td>
		</tr>
		<tr>
			<td>rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>00457</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>V900050</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>V900266</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan flash instruments</td>
			<td>Thermo Scientific</td>
			<td>IC100E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bongkrekate</td>
			<td>Biovision</td>
			<td>1820-100</td>
			<td></td>
		</tr>
		<tr>
			<td>atractyloside</td>
			<td>Sigma-Aldrich</td>
			<td>C4992</td>
			<td></td>
		</tr>
		<tr>
			<td>high-glucose Dulbecco&#8217;s modified Eagle medium</td>
			<td>Hyclone</td>
			<td>SH30022</td>
			<td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SV30087</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Invitrogen</td>
			<td>11860-038</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Thermo Scientific</td>
			<td>NCI3225CH</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

A produção de ATP por fosforilação oxidativa é a principal função das mitocôndrias. Mitocôndrias em eucariontes superiores também participam citosólico Ca2 + buffer, e a produção de ATP em mitocondrial pode ser mediada por intramitocondrial grátis Ca2 + concentração. Capacidade de retenção do CA2 + pode ser considerada como a capacidade das mitocôndrias de reter cálcio na matriz mitocondrial. Ca2 + pistas intracelulares acumuladas para a permeabilidade da membrana mitocondrial interna, denominado a abertura do poro de transição de permeabilidade mitocondrial (mPTP), menos de 1,5 kDa de peso que leva para o escapamento de moléculas com um molecular. CA2 +-desencadeada mitocôndrias inchaço é usado para indicar a abertura do mPTP. Aqui, descrevemos dois ensaios para examinar a capacidade de retenção do Ca2 + e Ca2 +-provocou inchaço mitocondrial em mitocôndrias isoladas. Depois de certas quantidades de Ca2 + são adicionados, todos os passos podem ser concluídos em um dia e gravados por um leitor de microplacas. Assim, estes dois ensaios simples e eficazes podem ser adoptados para avaliar a Ca2 +-relacionado funções mitocondriais.

Resultados de representante de Ca2 + capacidade de retenção: Os resultados foram expressos em valores de fluorescência. Com os pulsos adicionais de Ca2 + (proteína mitocondrial nmols/mg 200), a fluorescência aumentada o dobro acima da linha de base com a abertura do mPTP. 5 μM Bongkrekate (BKA), um inibidor de Ca2 +-induzida mPTP abertura, ou 1 μM atractyloside (ATR), um ativador de Ca2 +-induzida mPTP abertura, foram adiciona...

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Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Mitocondrial Ca2 + retenção capacidade do ensaio e Ca2 +-provocou inchaço mitocondrial do ensaio

Este protocolo destina-se a descrever um método para examinar a capacidade de retenção do Ca2 + e Ca2 +- desencadeada mitocondrial inchaço das mitocôndrias isoladas de SH-SY5Y células passo a passo.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

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Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

11.1K visualizações.  Qilu Hospital of Shandong University. Este protocolo destina-se a descrever um método para examinar a capacidade de retenção do Ca2 + e Ca2 +- desencadeada mitocondrial inchaço das mitocôndrias isoladas de SH-SY5Y células passo a passo.

Vídeo: Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Visualização de replicação do DNA mitocondrial em células individuais por Edu amplificação de sinal

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Neurociência

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Determinação de Membrana Mitocondrial Espécies de Oxigênio Reativas Potencial e em neurônios de rato vivo Cortical

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Métodos de Ensaio para Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

Avaliação da função bioenergética em células endoteliais Vascular Cerebral

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analisando o Transporte Mitocondrial e a Morfologia em neurônios derivados de células-tronco pluripotentes induzidos por humanos em Paraplegia Espástica Hereditária

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Geração de Organoides cerebrais humanos para modelagem de doenças mitocondriais

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Determinação da respiração mitocondrial e glicolise em amostras de tecido retinal ex vivo

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imagem Mitocondrial Ca2+ Absorção em Astrócitos e Neurônios usando Indicadores Ca2+ Codificados Geneticamente (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...