Pesquisas nos laboratórios é financiado pela Fundação Fritz-Bender, Munique, Alemanha (para TD) e da Universidade de Witten / Herdecke bolsa de investigação interna (para W.-KL). Gostaríamos de agradecer a Prof Dr. Dr. Frank Thévenod (Instituto de Fisiologia, Fisiopatologia e Toxicologia da Universidade de Witten / Herdecke) para sugestões.

1. Preparação dos Reagentes e Soluções <ol><li> Prepare uma solução de Hank equilibrada modificado sal (HBSS) (em mM: 136.9 NaCl, 5.37 KCl, 0,34 de Na 2 HPO 4, 0,44 KH 2 PO 4, 4,17 NaHCO 3, pH 7,2, sem vermelho de fenol). Armazenar a 4 ° C. </li><li> Adicione 1,5 M de CaCl2 e 2 M de 4- (2-hidroxietil) -1-piperazinoetanossulfónico (HEPES) (pH 7,2) soluções de reserva, utilizando água destilada. </li><li> Prepara-se uma solução de estoque de 250 mM de probenecida. Para a forma de ácido livre (muitas vezes referida como insolúveis em água), dissolver em pó probenecida em NaOH 1 N, encher a três quartos do volume final com água destilada e, lentamente, titula-se a pH 7,4 com 1 N de HCl. Armazenar a 4 ° C. </li><li> Dissolver membrana permeável ao cálcio fluorescente corante obrigatório em dimetilsulfóxido anidro (DMSO) a uma concentração de reserva de 1 mM. Armazenar a -20 ° C no escuro. </li><li> Antes de cada experiência, acabado de preparar-H contendo probenecidBSS de fluxo (PHF) tampão modificado pela combinação com HBSS (em concentrações finais) 1,5 mM de CaCl2, 10 mM de HEPES, pH 7,2, 5% de soro fetal bovino, 2 mM de probenecida, glicose e 5,55 mM. Aquece-se até 37 ° C. </li></ol> 2. Cultura celular <ol><li> Placa de 2,5 x 10 5 células de rim do túbulo proximal (WKPT CL.2-0293) por poço de uma placa de 6 ou 35 milímetros bem prato em meio de cultura padrão e crescer durante 2 dias, atingindo uma confluência de aproximadamente 70%. </li></ol> 3. Corante de cálcio Carregando <ol><li> Para cada prato ou bem, misturar 1 ml de meio de cultura contendo 5% de soro fetal bovino, corante de cálcio permeáveis ​​4 uM de ligação celular e 2 mM de probenecida. </li><li> Substituir o meio de cultura com 1 ml de mistura de corante de carga em cada poço e incubar a 37 ° C durante 30 min numa incubadora humidificada com 5% de CO 2. Cobrir a placa com uma folha de alumínio para evitar a descoloração do indicador fluorescente. </li></ol> 4. Preparação de Cells para Citometria de Fluxo <ol><li> Solução de tripsina a 0,05% de ácido etilenodiaminotetracético-quente (EDTA) a 37 ° C. </li><li> Aspirar cálcio solução de corante de carga de cada poço e adicionar 1 ml de tripsina ao longo da parede do poço. </li><li> Incubar a 37 ° C até que todas as células são separadas. </li><li> Transferência de células para um tubo de 1,5 ml e sedimento por centrifugação numa microcentrífuga a 10000 g durante 1 min. </li><li> Aspirar cuidadosamente o sobrenadante sem perturbar o sedimento. </li><li> Lave as células adicionando tampão PHF 1 ml e ressuspender por pipetagem cima e para baixo. </li><li> Sedimentar as células por centrifugação a 10.000 g durante 1 min. </li><li> Repita os passos 4.6. e 4.7. mais duas vezes. </li><li> Ressuspender as células num volume final de 500 ul de tampão de PHF e usar as células para a experimentação dentro de 1 h. </li><li> Opcional: contagem de células para determinar a concentração de células e usar 5 x outubro 4-1 x 10 5 células por medição. </li></ol> 5. Fluxo CitSetup ometer <ol><li> Comece o citômetro de fluxo, abra a gaveta fluídica e virar a chave seletora de válvula de ventilação para aumentar a pressão de ar do tanque de fluido do invólucro. </li><li> &quot;Prime&quot; do instrumento para remover eventuais bolhas de ar residente de dentro das câmaras de ar e câmara de fluxo. </li><li> No software de citometria de fluxo, abrir duas janelas do gráfico de pontos. <ol><li> O primeiro gráfico de pontos requer a frente de luz difusa (FSC) para o parâmetro X e do lado da luz difusa (SSC) para o parâmetro Y para controlar para a qualidade das células no interior da amostra através do seu tamanho e da sua granularidade, respectivamente. </li><li> O segundo gráfico de pontos mede a intensidade de fluorescência do corante ligado de cálcio. Escolha do canal de detecção de fluorescência apropriado para o parâmetro Y utilizando uma escala logarítmica e tempo em segundos para o parâmetro X em uma escala linear. </li></ol></li><li> Definir a taxa de fluxo para &quot;alto&quot;. </li></ol> 6. Citometria de Fluxo MEDIÇÃO <ol><li> Execute as medições em RT. Num tubo de ensaio de citometria de fluxo (11,5 x 75 mm), adicionar 480 ul de tampão de PHF. </li><li> Adicionar suspensão de células de 20 mL e brevemente vortex para misturar. </li><li> Mover o braço de suporte do tubo para o lado e posicionar o tubo de amostra sobre o tubo de injecção da amostra até uma vedação hermética é formada. Retorne o braço de suporte do tubo para sua posição original centrado. </li><li> Optimizar a medição, ajustando o canal de fluorescência de tal modo que a intensidade de fluorescência média da amostra é de aproximadamente 10 2 no eixo-y. Salvar e usar para os experimentos posteriores. </li><li> Para iniciar um experimento, repita os passos 6.1 a 6.3. </li><li> Fluorescência basal recorde para 50 seg. </li><li> Pause medida, retirar o tubo de amostra, adicionar o composto de interesse, vortex brevemente e retornar ao tubo de injecção de amostra. Este passo não deve demorar mais do que 15 segundos. </li><li> Retomar a medição e continuar para um total de 204,8 seg. </li><li> Use um i cálcioonophore, tais como ionomicina (10 uM), e um quelante de cálcio, tal como o etileno-glicol do ácido tetra-acético (EGTA), MnCl2 ou CoCl2 (2 mM), para os controlos positivos e negativos, respectivamente. </li></ol> Análise 7. Os dados <ol><li> Analisar dados usando o software de citometria de fluxo dedicado para análise off-line, como WinMDI (Windows Multiple Document Interface para Citometria de Fluxo), um pacote de software livre desenvolvido por Joe Trotter no Instituto Scripps, Citometria de Fluxo Núcleo Facility. </li><li> Abra um gráfico de pontos com os parâmetros SSC e FSC. </li><li> Selecione a região de células para análise utilizando a ferramenta &quot;Regiões&quot; (clique com o botão direito sobre a imagem) para excluir as células mortas ou restos de células encontradas no canto inferior esquerdo da análise dos dados (Figura 1). </li><li> Análise quadrante (Figura 2) <ol><li> Abra uma densidade de trama com o tempo no eixo dos x e intensidade de fluorescência no eixo-y. Use todos os eventos. </li><li> NosE é a mesma região gating como em 7.3. </li><li> Quantificar os dados usando a ferramenta &quot;Quadrante&quot;. </li><li> Ajustar os separadores de quadrante, de modo que a linha vertical é colocado no momento da adição do composto e a linha horizontal que está colocado no centro da linha de base de fluorescência nos controlos. Certifique-se de que o posicionamento dos separadores de quadrante é igual em todas as amostras. </li><li> A percentagem de células em cada quadrante é apresentado em cada canto (Figura 2). Compare o quadrante superior direito (UR) entre as amostras. </li></ol></li><li> Análise de histograma (Figura 3) <ol><li> Abra os dados de controlo de uma janela do histograma de intensidade de fluorescência no eixo dos x e eventos no eixo-y. Use todos os eventos. </li><li> Para uma visão pictórica, adicione os dados de teste para ser comparado com a testemunha como parcelas de sobreposição (vá em File → Open File → Selecione o arquivo → Overlay). </li><li> Para a análise quantitativa, único histograma ganhardows deve ser usado. Portão da população celular utilizando a região R1 do passo 7.3. </li><li> No controlo não-tratadas, usar a função de marcador (&quot;marcadores&quot;) para marcar a área de análise a partir do ponto médio entre o mínimo e máximo de fluorescência do pico de controlo e terminando na intensidade de fluorescência máxima. A percentagem de células nessa área é calculada. </li><li> Recuperar os dados da janela de estatísticas. Repita o procedimento para os restantes ficheiros de dados usando a mesma área marcada para análise. </li></ol></li><li> Análise da intensidade de fluorescência (Figura 4) <ol><li> Abrir um gráfico de pontos com o tempo no eixo dos x e intensidade de fluorescência no eixo-y. Use todos os eventos. </li><li> Ative o &quot;Modo Kinetics&quot; e &quot;Overlay Kinetics Line&quot;. Uma linha azul aparecerá na trama ponto que representa a intensidade média de fluorescência. </li><li> Quantificar o fluxo relativo de cálcio através da criação de várias regiões retangulares. Cada região deve ter awidth de cerca de &quot;50&quot;, o que é equivalente a &quot;10 seg&quot;. Defina até 15 regiões retangulares. </li><li> Recuperar quantificação da janela de estatísticas e copiar em uma planilha. </li><li> Usar os dados-y significativo para análise. Calcule a média de R2 a R6, que é equivalente a 20 a 50 seg. Este é o valor da linha de base. </li><li> Escolha um intervalo de tempo adequado para a análise do influxo de cálcio (depende de início, duração e intensidade). </li><li> Calcular os sinais de cálcio a partir de cada região, dentro deste intervalo de tempo em relação ao valor da linha de base média, isto é, o sinal de fluorescência antes da adição de composto, a qual é definida como 100%. Determinar a média eo desvio padrão das regiões postar além composto; este é o sinal significativo de cálcio evocado pelo composto. </li></ol></li></ol>

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Brini, M., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+pumps+in+health+and+disease.">Calcium pumps in health and disease.</a> Physiol Rev. 89, 1341-1378 (2009).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+trisphosphate+and+calcium+signalling.">Inositol trisphosphate and calcium signalling.</a> Nature. 361, 315-325 (1993).</li><li>Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+actions+of+calcium-mobilizing+messenger,+inositol+1,4,5-trisphosphate.">Formation and actions of calcium-mobilizing messenger, inositol 1,4,5-trisphosphate.</a> Am J Physiol. 252, 149-157 (1987).</li><li>Putney, J. W., Ribeiro, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+between+the+plasma+membrane+and+endoplasmic+reticulum+calcium+stores.">Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores.</a> Cell Mol Life Sci. 57, 1272-1286 (2000).</li><li>Soboloff, J., Rothberg, B. S., Madesh, M., Gill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STIM+proteins:+dynamic+calcium+signal+transducers.">STIM proteins: dynamic calcium signal transducers.</a> Nat Rev Mol Cell Biol. 13, 549-565 (2012).</li><li>Williams, G. S., Boyman, L., Chikando, A. C., Khairallah, R. J., Lederer, W. J. <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+uptake.">Mitochondrial calcium uptake.</a> Proc Natl Acad Sci U S A. 110, 10479-10486 (2013).</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, 335-344 (2009).</li><li>Monteith, G. R., Davis, F. M., Roberts-Thomson, S. 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+channels+and+pumps+in+cancer:+changes+and+consequences.">Calcium channels and pumps in cancer: changes and consequences.</a> J Biol Chem. 287, 31666-31673 (2012).</li><li>Roderick, H. 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=Calcium+in+the+heart:+when+it's+good,+it's+very+very+good,+but+when+it's+bad,+it's+horrid.">Calcium in the heart: when it's good, it's very very good, but when it's bad, it's horrid.</a> Biochem Soc Trans. 35, 957-961 (2007).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260, 3440-3450 (1985).</li><li>Jin, X., 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=Cilioplasm+is+a+cellular+compartment+for+calcium+signaling+in+response+to+mechanical+and+chemical+stimuli.">Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli.</a> Cell Mol Life Sci. , (2013).</li><li>Valet, G., Raffael, A., Russmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+calcium+in+vital+cells+by+flow-cytometry.">Determination of intracellular calcium in vital cells by flow-cytometry.</a> Naturwissenschaften. 72, 600-602 (1985).</li><li>June, C. H., Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+cellular+ionized+calcium+concentration.">Flow cytometric measurement of cellular ionized calcium concentration.</a> Pathology and immunopathology research. 7, 409-432 (1988).</li><li>Ransom, J. T., DiGiusto, D. L., Cambier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+intracellular+calcium+mobilization.">Flow cytometric analysis of intracellular calcium mobilization.</a> Methods Enzymol. 141, 53-63 (1987).</li><li>Gergely, L., Cook, L., Agnello, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+Ca2++flux+measurement+on+isolated+human+B+cells+that+uses+flow+cytometry.">A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.</a> Clin Diagn Lab Immunol. 4, 70-74 (1997).</li><li>Seidel, J., 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=The+neurotransmitter+GABA+is+a+potent+inhibitor+of+the+stromal+cell-derived+factor-1alpha+induced+migration+of+adult+CD133++hematopoietic+stem+and+progenitor+cells.">The neurotransmitter GABA is a potent inhibitor of the stromal cell-derived factor-1alpha induced migration of adult CD133+ hematopoietic stem and progenitor cells.</a> Stem cells and development. 16, 827-836 (2007).</li><li>Lee, W. K., Chakraborty, P. K., Roussa, E., Wolff, N. A., Th&#233;venod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK1/2-dependent+bestrophin-3+expression+prevents+ER-stress-induced+cell+death+in+renal+epithelial+cells+by+reducing+CHOP.">ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.</a> Biochim Biophys Acta. 1823, 1864-1876 (2012).</li><li>Woost, P. 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=Immortalization+and+characterization+of+proximal+tubule+cells+derived+from+kidneys+of+spontaneously+hypertensive+and+normotensive+rats.">Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats.</a> Kidney Int. 50, 125-134 (1996).</li><li>Beyer, K. H., 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=Benemid,'+p-(di-n-propylsulfamyl)-benzoic+acid;+its+renal+affinity+and+its+elimination.">Benemid,' p-(di-n-propylsulfamyl)-benzoic acid; its renal affinity and its elimination.</a> Am J Physiol. 166, 625-640 (1951).</li><li>Buckley, B. J., Whorton, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunicamycin+increases+intracellular+calcium+levels+in+bovine+aortic+endothelial+cells.">Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells.</a> Am J Physiol. 273, 1298-1305 (1997).</li><li>Koepsell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SLC22+family+with+transporters+of+organic+cations,+anions+and+zwitterions.">The SLC22 family with transporters of organic cations, anions and zwitterions.</a> Molecular aspects of medicine. 34, 413-435 (2013).</li><li>Dean, M., Hamon, Y., Chimini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+ATP-binding+cassette+(ABC)+transporter+superfamily.">The human ATP-binding cassette (ABC) transporter superfamily.</a> J Lipid Res. 42, 1007-1017 (2001).</li><li>Brezden, C. B., Hedley, D. W., Rauth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+expression+of+P-glycoprotein+as+a+determinant+of+loading+with+fluorescent+calcium+probes.">Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes.</a> Cytometry. 17, 343-348 (1994).</li><li>Th&#233;venod, F., Friedmann, J. M., Katsen, A. D., Hauser, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+multidrug+resistance+P-glycoprotein+via+nuclear+factor-kappaB+activation+protects+kidney+proximal+tubule+cells+from+cadmium-+and+reactive+oxygen+species-induced+apoptosis.">Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis.</a> J Biol Chem. 275, 1887-1896 (2000).</li><li>Chakraborty, P. K., Lee, W. K., Molitor, M., Wolff, N. A., Th&#233;venod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadmium+induces+Wnt+signaling+to+upregulate+proliferation+and+survival+genes+in+sub-confluent+kidney+proximal+tubule+cells.">Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells.</a> Mol Cancer. 9, 102 (2010).</li><li>Jennings, L. K., Dockter, M. E., Wall, C. D., Fox, C. F., Kennedy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+mobilization+in+human+platelets+using+indo-1+and+flow+cytometry.">Calcium mobilization in human platelets using indo-1 and flow cytometry.</a> Blood. 74, 2674-2680 (1989).</li><li>Lopez, M., Olive, D., Mannoni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cytosolic+ionized+calcium+variation+in+polymorphonuclear+leukocytes+using+flow+cytometry+and+Indo-1+AM.">Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo-1 AM.</a> Cytometry. 10, 165-173 (1989).</li></ol>

The authors have nothing to disclose.

O cálcio é um evento chave na múltiplos processos celulares que actuam como um segundo mensageiro molécula de sinalização e também como um meio para comunicar entre o ER e mitocôndrias. A capacidade de medir alterações nas concentrações de cálcio citosólico livre é uma técnica útil que pode ser aplicada a todas as áreas de biologia celular. Existem vários métodos para a detecção de sinais de cálcio citosólicos. Classicamente, os sinais de um indicador de cálcio raciométrica, tais como Fura-2, s...

O cálcio é um importante segundo mensageiro responsáveis ​​pela transmissão de sinais fisiológicos e patofisiológicos celular 1. Concentrações citosólicas de cálcio são mantidos baixos por meio da atividade de bombas de cálcio e trocadores de cálcio. A / ATPase de cálcio do retículo endoplasmático sarcoplasmático (SERCA) preenche o retículo endoplasmático (ER) loja de cálcio como parte de um sistema de &quot;vazamento de bomba&quot;, enquanto o cálcio ATPase da membrana plasmática (PMCA) expulsa o cálcio para o compartimento extracelular, tanto em maneiras dependentes de ATP 2. Mensageiros fisiológicas, tais como hormonas ou neurotransmissores, transmitem os seus sinais através de receptores acoplados à proteína G, a activação de fosfolipase C, que provoca a hidrólise de fosfatidilinositol 4,5-bifosfato (PIP2) na membrana de plasma para gerar diacilglicerol e trifosfato de inositol (IP3) 3. Enquanto diacilglicerol permanece na membrana plasmática, IP3 difunde-se para o citosol e liga-se ao receptor de IP3s (IP3Rs), que são activadas ligando os canais de cálcio, encontrados na membrana do RE e do cálcio é libertado a partir da loja ER luminal culminando com um aumento nas concentrações de cálcio citosólicos 4. Uma via alternativa para o cálcio para atingir o citossol é através de canais de cálcio e permutadores presentes na membrana plasmática. Crosstalk entre esses dois compartimentos foram descritos: liberação de cálcio de cálcio induzida (CICV), onde o cálcio extracelular induz liberação de cálcio das lojas ER 5 e loja operada entrada de cálcio (SOCE), onde o esvaziamento das lojas ER é detectada por STIM e provoca a abertura de Orai canais de cálcio na membrana plasmática para promover o reenchimento das lojas de 6 ER. Sob condições fisiopatológicas, a resposta celular de cálcio é desregulados e citosólicas de cálcio aumenta na ausência de estimuladores fisiológicos 1. A resposta de cálcio pode ser afectada de várias maneiras: cálcio prolongadasinal, a remoção retardada de cálcio a partir do citosol, depleção das reservas de cálcio ou mudanças localizadas em cálcio. Além disso, as mitocôndrias assumir um papel central na memória intermédia de libertação de cálcio e 7. Prolongado e / ou aumento de cálcio citosólico supramáxima leva à morte celular. Na verdade, o cálcio é mais frequentemente do que não envolvido na resposta fisiopatológica celular e é um evento chave em uma ampla gama de doenças, tais como doenças neurodegenerativas, doenças cardíacas, câncer, doenças ósseas e doenças renais, que estão principalmente associados com a morte celular e perda ou alteração de órgão ou tecido função 8-10. Além disso, a perturbação de sinais de cálcio tem sido associada a adaptação celular e alterações em funções celulares e as respostas. Tradicionalmente, os sinais de cálcio são medidos com um indicador de cálcio fluorescente carregada negativamente que é carregado em células em uma forma de éster de acetoximetilo permeável célula (AM). Uma vez clivada por Esteras celulareses, os indicadores fluorescentes permanecer dentro do compartimento intracelular e aumentam em intensidade de fluorescência quando o cálcio é ligado. O indicador de cálcio mais conhecidos e utilizados é o raciométrica Fura-2 desenvolvido por Roger Tsien e colegas 11. Indicadores de cálcio com afinidades diferentes para permitir diferentes pools de cálcio de cálcio a ser monitorizado. Métodos de detecção incluem imagens de células vivas, leitor de microplacas de fluorescência e citometria de fluxo. A cinética relativamente rápida de sinais de cálcio (geralmente dentro de alguns segundos a minutos) faz de células vivas imaginando o melhor método para a obtenção de mais informações sobre as características do sinal de cálcio. Além dos cinética rápida de sinais de cálcio (em milissegundos), imagens de células vivas é um método melhor para estudar compartimentalização celular da sinalização de cálcio dentro de uma única célula 12. As concentrações de cálcio absolutos pode ser calculada através da determinação da fluorescência mínima e máxima da ind cálcioicator pela adição de um quelante de cálcio e de permeabilização das células, respectivamente, como descrito por Grynkiewicz et ai. 11. A utilização de citometria de fluxo para medir sinais de cálcio foi desenvolvido pela primeira vez na década de 1980 em células imunológicas onde a oportunidade para medir vários parâmetros, tais como a integridade da membrana e separação de população de células, e o requisito de células em suspensão combinada com o desenvolvimento de um único comprimento de onda indicadores de cálcio feita de citometria de fluxo de um método ideal e convenientdetection 13-15. Em células aderentes, imagens de células vivas fornece a maior parte da informação sobre a sinalização de cálcio, mais importante, a cinética, mas requer uma instalação complicada, compreendendo um microscópio de fluorescência, um sistema de perfusão, a manutenção do ambiente celular, tais como a temperatura, e microscópio software especializado para adquirir e análise de dados. Métodos alternativos, tais como um leitor de microplacas de fluorescência ou farmacolmeios ogical através do uso de quelantes de cálcio são incomparáveis ​​em termos de informação adquirida. Citometria de fluxo é cada vez mais amplamente utilizado para monitorizar o cálcio citosólico em células aderentes, embora, na maioria dos estudos, apenas ponto de terminar as medições sejam efectuadas. Utilizando o método desenvolvido inicialmente por Gergely et al., Em células imunológicas B 16, alterações na concentração de cálcio livre citosólico por citometria de fluxo com uma cinética em tempo real e o controlo da intensidade de fluorescência em células individuais a partir de uma população de amostra foram medidos com sucesso em células epiteliais de cancro e de células-tronco do câncer híbridos 17. Este método foi adicionalmente adaptado para utilização em células epiteliais aderentes, que são difíceis de carregar com os indicadores de cálcio 18. Aqui, utilizando uma linha de células epiteliais aderentes imortalizada derivada do segmento S1 do túbulo proximal do rim de rato (WKPT-CL.2 0293) 19, descrevemos um m simplificada optimizadoétodo para a medição de alterações na concentração de cálcio citosólico, por citometria de fluxo. Uma vez que muitas células epiteliais possuem um número de transportadores orgânicos para compostos aniónicos, a carga de células com um indicador de cálcio podem provar ser um desafio. Para evitar o efluxo de indicadores de cálcio, probenecida, o inibidor prototípico de transportadores de aniões orgânicos e originalmente desenvolvido para diminuir a excreção renal de penicilina 20, é aplicado simultaneamente no meio de incubação. As células são mantidas em monocamadas de carga indicador de cálcio, apresentado em sinais de suspensão e de cálcio podem ser detectadas imediatamente após a adição do composto.

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:79px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:321px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-3, AM</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">F-1241&#160;</td>
			<td>Cell permeable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Probenecid</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8761</td>
			<td>Water insoluble, dissolve in 1 N NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ionomycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thapsigargin</td>
			<td style="width:79px;">Tocris Bioscience</td>
			<td style="width:119px;">1138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tunicamycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7765</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCalibur Flow Cytometer + CELLQuest software</td>
			<td style="width:79px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td>
			<td style="width:79px;">Joe Trotter, The Scripps Institute</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td>
			<td style="width:79px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made available by Dr. Ulrich Hopfer (Department of Physiology &#38; Biophysics, Case 
			Western Reserve University, Cleveland, OH)</td>
		</tr>
	</tbody>
</table>

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.

Para aumentar o cálcio citosólico, dois compostos farmacológicos foram aplicados a células renais do túbulo proximal (WKPT-0293 CL.2) carregadas com o corante de ligação celular permeável ao cálcio, Fluo-3-AM. Amostras de controlo não tratados foram carregadas com corante de ligação do cálcio na presença de probenecida e foram misturados, mas sem a adição de quaisquer compostos. Tapsigargina (TG) é um inibidor da bomba clássica SERCA que conduz a fugas para fora do ER e resultando em aumento de cálcio...

Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

O cálcio está envolvido em inúmeras vias de sinalização fisiológicos e fisiopatológicos. Imagem de células vivas requer equipamento especializado e pode ser demorado. Um método rápido, simples usando um citômetro de fluxo para determinar mudanças relativas de cálcio citosólico em células epiteliais aderentes trazidos para suspensão foi otimizado.

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 ...

cytosolic-calcium-measurements-renal-epithelial-cells-flow

Research

JoVE Journal

Biology

15.2K visualizações.  University of Witten/Herdecke.  O cálcio está envolvido em inúmeras vias de sinalização fisiológicos e fisiopatológicos. Imagem de células vivas requer equipamento especializado e pode ser demorado. Um método rápido, simples usando um citômetro de fluxo para determinar mudanças relativas de cálcio citosólico em células epiteliais aderentes trazidos para suspensão foi otimizado. 

Vídeo: Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

FCM-IP: Imunoprecipitação Detectado por citometria de fluxo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Biologia

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificação de Citometria de Fluxo de células de camundongo meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Diferenciação de alta eficiência de pluripotentes humanas células-tronco para Cardiomyocytes e Caracterização por citometria de fluxo

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicação por citometria de fluxo de protoplastas de tubérculos isolados de medição

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Medição do elevado-throughput de Dictyostelium discoideum macropinocitose por citometria de fluxo

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Detectando e caracterizando a auto-montagem da proteína in vivo por citometria de fluxo

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Medição da massa mitocondrial e potencial de membrana em células-tronco hematopoiéticas e células T por citometria flow

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...