Este trabalho foi apoiado pelo prêmio American Heart Association 13SDG14500046 para PD, uma Educação Energia Sustentável &amp; Centro de Pesquisa Award (Univ. of Tennessee) para PD, e de Educação Médica dos Médicos e Fundação de Pesquisa (Univ. of Tennessee) prêmio para JM A autores agradecem Caroline Leplante (Yale Univ.) para o protocolo para a conversão de levedura para esferoplastos; Eric T. Boder (Univ. of Tennessee) para o uso de suas incubadoras agitação, mesa centrífuga e Western Blot equipamentos de análise, e do Centro de Biotecnologia Ambiental (Univ. Tennessee) para o uso de seu ultra-centrífuga; Günther Daum para anticorpos levedura (Graz Univ of Technology, na Áustria.); o pessoal do Departamento de Obstetrícia e Ginecologia (Univ. Tennessee Medical Center) para assistência técnica .

1. Isolando lipídicas gotas de (cisão) Células de levedura Isolamento de gotículas do organismo modelo popular brotamento Saccharomyces cerevisiae é quase idêntico ao seguinte protocolo 6. As diferenças nas preparações são anotados. 1. Crescer células de levedura <ol><li> Prepare a mídia. Combine 36 g de YE5S pó por litro de dH 2 O em garrafas de vidro ou frascos de cultura. Serão necessários cerca de 2 L de mídia. Autoclave a mídia a 121 ° C por 20 min. Permita que os meios de comunicação para arrefecer à temperatura ambiente. Para S. cerevisiae substituir YE5S com YPD. </li><li> Coloque 10-20 ml dos meios de comunicação YE5S resfriado em uma garrafa de cultura de 250 ml, utilizando técnicas estéreis. Inocular os meios de comunicação com uma pequena quantidade de células de levedura de uma placa de ágar utilizando uma vara de madeira estéril ou equivalente. Deixe as células crescer até à densidade óptica pretendida, a 30 ° C. Medir a densidade óptica a umcomprimento de onda de 595 nm utilizando um espectrofotómetro. </li><li> Coloque a mídia restante nos frascos de 2,8 L. Usar não mais do que 1 L de balão por meios de 2,8 L para assegurar uma mistura adequada durante o crescimento celular na incubadora de agitação. O rendimento final de células molhadas deve ser cerca de 10 g para ser capaz de adquirir gotículas lipídicas suficientes para análise proteômica e lipidomas. </li><li> Introduzir nas células do passo 1.2 em balões 2,8 L, tendo cada um 1 L de meios que foram preparados no passo 1.1. </li><li> Crescer as células com a densidade desejada, a 30 ° C. </li><li> Certifique-se que as células têm gotículas lipídicas. Reserva 1 ml das células. Medir a densidade óptica (OD) da amostra. Adicionar 0,1 ml de um estoque de 100 mM de BODIPY 493/503, em etanol por OD de células na amostra. Colocar 3 mL da amostra entre uma lâmina de vidro e uma lamela de vidro. Visualizar sob um microscópio fluorescente com um filtro de GFP (Figura 1A). </li><li> Sedimentar as células do passo 1.5 a 4 &amp; #176; C a 3.800 g durante 10 min. Trabalhar em lotes de acordo com a capacidade dos tubos de centrífuga. </li><li> Deitar fora a mídia YE5S e lavar as células com um tampão de EMM + 600 mM sorbitol. Sorbitol fornece suporte osmótico. </li><li> Transferência das células para um tubo de centrífuga de 50 ml estéril e sedimentar as células a 4 ° C a 2.000 g durante 10 min. Remover o sobrenadante. Pesar as células húmidas. Ressuspender as células em 2 ml de EMM (+ sorbitol 600 mM) de tampão por grama de células. </li></ol> 2. A conversão de células de levedura de fissão em esferoplastos e subsequente lise <ol><li> Adicionam-se 5 mg cada um de 1) enzima lítica de levedura e 2) de levedura de lise enzimas do fungo Trichoderma harzianum por grama de células húmidas, que foram pesadas no passo 1.9. Para S. cerevisiae aumentar estas enzimas com a mesma quantidade de Zymolyase. </li><li> Incubar as células a 30 ° C, sob agitação suave a 100 rpm durante 60 min. </li><li> Certifique-se de que os esferoplastos têm dr lipídicooplets. Ao repetir o passo 1.6 (Figura 1B). </li></ol> 3. Densidade centrifugação em gradiente <ol><li> Agregar as esferoplastos por centrifugação a 4 ° C a 2.000 g durante 5 min. </li><li> Remova cuidadosamente o sobrenadante com uma pipeta de transferência de plástico. </li><li> Suavemente ressuspender os esferoplastos peletizadas com gelo frio de 10 mM Tris-HCl, 600 mM de sorbitol, e 200 ^ M de EDTA. Tenha em mente que os esferoplastos são muito frágeis. </li><li> Agregar as esferoplastos novamente usando as mesmas configurações como no passo 3.1. Remover cuidadosamente o sobrenadante e ressuspender as esferoplastos utilizando uma pipeta de transferência de plástico em 12% de Ficoll, 10 mM Tris-HCl, e 200 ^ M de EDTA a uma concentração de 2 ml / g de células. </li><li> Transferir os esferoplastos em suspensão para um homogeneizador de vidro e homogeneizar em gelo, 20-30 pancadas, com o pilão solto do homogeneizador de vidro. </li><li> Transfira os esferoplastos lisadas para tubos de centrífuga e sobreposição com o mesmo volume de 12% de Ficoll, 10 mM Tris-HCl, tampão e 200 fiM de EDTA. Escolha o número de tubos de centrífuga de usar para que cada tubo é de cerca de dois terços cheio. </li><li> Centrifugar a 4 ° C a 100,000 xg durante 1,5 horas num rotor SW28 ou equivalente. Permitir rotor a costa a uma parada (rotor desaceleração = 0). </li><li> Suavemente transferir a camada sobrenadante branco (Figura 2A), que contém as gotas, para um novo tubo (s) de centrifugação usando uma pipeta ou uma pipeta Pasteur curvada. Adicionar um volume suficiente de 12% de Ficoll, 10 mM Tris-HCl, tampão de EDTA e 200 ^ M para a amostra de modo que ele preenche mais ou menos um terço do tubo de centrifugação. </li><li> Adicionar o volume equivalente de 8% de Ficoll, 10 mM Tris-HCl, tampão de EDTA e 200 ^ M para o topo da amostra (s) no novo tubo (s) de centrifugação. Após a adição desta amortecer os tubos devem ser de cerca de dois terços do volume. </li><li> Centrifugar a 4 ° C a 100,000 xg durante 1 hora num rotor SW28 ou equivalente. Permitir rotor a costa a uma parada (rotor desaceleração = 0). </li><li> Suavemente transferir a camada de topo branco flutuante (Figura 2B), que irá conter as gotículas, para um novo tubo (s) de centrifugação usando uma pipeta ou uma pipeta Pasteur curvada. Adicionar 600 mM de sorbitol, 8% de Ficoll, 10 mM Tris-HCl, tampão de EDTA e 200 ^ M para a amostra de modo a que ocupe cerca de um terço do tubo de centrifugação. </li><li> Adicionar o volume equivalente a 250 mM de sorbitol, 10 mM Tris-HCl, e tampão EDTA 200 uM para o topo da amostra (s) no novo tubo (s) de centrifugação. Após a adição desta amortecer os tubos devem ser de cerca de dois terços do volume. </li><li> Centrifugar a 4 ° C a 100,000 xg durante 1 hora num rotor SW28 ou equivalente. Permitir rotor a costa a uma parada (rotor desaceleração = 0). </li><li> Transferir parte superior da camada branca (Figura 2C), que deve conter apenas as gotículas de lípidos e proteínas ligadas, a tubagem de diálise, e diálise O / N em 10 mM Tris-HCl e 200 ^ M de tampão de EDTA para eliminar o Ficoll. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Isolando lipídicas Gotas de placenta humana vilosidades Células Placentas foram coletadas de mulheres saudáveis ​​com gestações únicas submetidos de forma eletiva entrega cesariana antes do início do trabalho de parto espontâneo no prazo. Assuntos deu o consentimento escrito e informado para a coleção de sua placenta. A coleção, e posterior utilização, de placentas foi realizada com a aprovação da Universidade do Tennessee e Universidade do Tennessee Graduate School of Medicine, em Knoxville Institutional Review Board (# 8757B e # 3338, respectivamente). 1. Isolamento de células das vilosidades da placenta humana Parte 1 do protocolo é uma modificação de um protocolo publicado anteriormente por 26 Petroff et al. Prepare as seguintes soluções: <ul><li> De NaCl (1 L): Dissolver 9 g de NaCl (M 58,4 g / mol) em dH 2 O, para se obter uma solução de 1 litro. Filtro de esterilizar (0,2; MM membrana). </li><li> Solução salina equilibrada de 10x Hank (10x HBSS) (1 L): 4 g de KCl (M 74,5 g / mol); de 0,6 g de KH 2 PO 4 (M 136,086 g / mol), 80 g de NaCl (M 58,44 g / mol); Na 2 HPO 4 (M 141,96 g / mol), 10 g de D-glucose (M 180,16 g / mol). Dissolve-se cada um dos componentes em dH 2 O, para se obter uma solução de 1 litro. Filtro de esterilizar (0,2 membrana mm). </li><li> Tampão de digestão de enzima (0,6 ml): combinar 70 ml 10x HBSS com 3,3 ml de 7,5% de Na Biscarbonate, 17,5 mL de HEPES 1 M, e 266,1 ml de dH2O; filtro de esterilizar (membrana de 0,2 um). Armazenar a 4 ° C. </li><li> DNase I: imediatamente antes da utilização, adicionar 5 ml estéril 0,9% de NaCl para um frasco de DNase. Mantenha no gelo até o uso ou armazenar a -20 ° C. </li></ul><ol><li> Preparando placenta para dissecção <ol><li> Obter uma placenta humana e processo tão perto do tempo de entrega possível. Usar um recipiente selado, tal como um refrigerador para transportar a placenta. Tenha cuidado ao ladoling materiais biológicos humanos. </li><li> Na capa de segurança biológica, transferir a placenta para um recipiente estéril autoclave, e lavar cuidadosamente o sangue a partir de placenta e de membranas utilizando solução estéril a 0,9% de NaCl (salina). Descarte sangrenta salina no 1 L proveta como lixo biológico líquido. </li><li> Coloque a placenta em um campo estéril, com a superfície lisa, tendo o cordão umbilical voltada para cima. Com nítidas, tesoura de ponta fina e pinças remover membranas fetais e do cordão umbilical. </li><li> Vire a placenta de forma que a superfície materna (superfície áspera) é voltado para cima. Remover o tecido sobreposta placa basal, cerca de 3 mm a partir da superfície. </li></ol></li><li> Dissecando a placenta <ol><li> Dissecar um cotilédone de cada vez evitando a placa coriônica. Colete tecido das vilosidades em um copo de 250 ml com solução salina. </li><li> Lavar tecido várias vezes em um copo separado com 0,9% de NaCl por agitação com uma pinça, utilizando o copo de 1 L para resíduos líquidos. </li><li> Transferir um cotilédone de cada vez para uma placa de Petri 150 milímetros. Segure com uma pinça e raspar tecido com lâmina de barbear de navios na placa de Petri. Colocar o tecido raspado num copo separado, contendo 0,9% de NaCl. Repita o procedimento para todas as peças de tecido. </li><li> Transferir pequena porção do tecido da peneira de dissociação de células e enxaguar com 0,9% de NaCl extensivamente até o eluato torna-se clara. Repetir para todo o tecido. </li><li> Depois de enxaguar o tecido raspado do crivo de dissociação celular, drenar o excesso líquido e peso. Neste ponto, o tecido pode ser armazenado S / N, num balão de Erlenmeyer estéril a 4 ° C em DMEM. </li></ol></li><li> A digestão enzimática do tecido placentário <ol><li> Preparar a mistura de digestão do tecido: 100 ml de tampão de diluição de enzima pré-aquecido, 1 ml de DNase I e 20 ml de 2,5% de tripsina 10x. </li><li> Dividir o tecido, a transferência de 60 g a partir de tecido recolhido total (geralmente cerca de 250-300 g) para um balão de Erlenmeyer esterilizado de 500 ml. </li><li&gt; Adicionar a mistura de digestão de tecido para o balão de Erlenmeyer contendo o tecido, e incubar a 37 ° C num agitador durante 45 minutos a 150 rpm. </li></ol></li><li> Coletar as células dissociadas <ol><li> Após os primeiros 45 min de dissociação, defina o recipiente de digestão em uma inclinação até que o tecido se instala. </li><li> Recolher o sobrenadante, tomando cuidado para não coletar tecido não dissociada. Adicionar uma quantidade igual de HBSS para o sobrenadante foi recolhido e transferir para tubos estéreis de 15 ml de centrífuga. Para porções adicionais de tecido não dissociado, repetir procedimento a partir do passo 3.2 com o tecido não dissociado. </li><li> Centrifuga-se o homogeneizado a 4 ° C a 1.000 g durante 15 min. </li><li> Para cada lote, após a centrifugação, aspirar o sobrenadante sem perturbar o sedimento. As células das vilosidades da placenta são predominantemente na parte branca da pelota, sobrepondo-se as células vermelhas do sangue. </li><li> Transferência das células das vilosidades (parte branca da pelota) para a sterile 50 ml tubo de centrifugação cónico e manter em gelo. </li><li> Depois de recolher as células a partir de todas as três fases de digestão, filtrar a suspensão através de um filtro de 100 mm de células de nylon inserida no topo de um tubo de centrífuga de 50 ml estéril cónica. Se a filtração da suspensão de células diminui, levante sobre o filtro para extrair um vácuo no interior do tubo. </li><li> Centrifugar a 4 ° C a 1.000 g durante 10 min. Remova cuidadosamente o sobrenadante sem perturbar o sedimento. </li></ol></li></ol> 2. Homogeneização de células das vilosidades da placenta <ol><li> Antes de iniciar o processo de isolamento, preparar 50 ml de lise hipotónico Médio (HLM) contendo 20 mM Tris-HCl, pH 7,4 e EDTA 1 mM. Adicionar os inibidores da protease de HLM imediatamente antes da utilização e manter o meio em gelo. </li><li> Na capa de segurança biológica, adicionar 4 vezes o volume do pelete celular HLM gelada para as células. Forma suave e profunda voltar a suspender as células por pipetagem as células-se-and-baixo com uma pipeta de 10 ml. </li><li> Incubar as células em suspensão em gelo durante 10 min. </li><li> Transferência das células para o homogeneizador de Dounce, a qual deve estar em gelo. Lentamente homogeneizar as células através da aplicação de 20-25 movimentos suaves com o pilão folgada do homogeneizador. </li><li> Centrifugar o lisado celular a 4 ° C a 3000 xg durante 10 min para remover as células não rompidas, núcleos e restos celulares. </li><li> Recolhe-se o sobrenadante e transferir para um tubo SW28 (ou uma alternativa que pode ser centrifugado a 25.000 x g). Centrifugar a 4 ° C a 25000 x g durante 20 min para remover as mitocôndrias. </li></ol> 3. Isolando gotículas lipídicas por ultracentrifugação <ol><li> Preparar 50 ml de carbonato de sódio 100 mM (M 106 g / mol) de tampão (pH 11,5) e 50 ml de solução a 60% (w / w) de sacarose. Adicionar os inibidores de protease para o tampão de carbonato de sódio imediatamente antes da utilização e manter em gelo. </li><li> Recolhe-se o sobrenadante a partir do passo 2.6 para um tubo de centrífuga de 50 ml, ajustara 20% de sacarose e de transferência para a parte inferior de um tubo de ultracentrífuga para o rotor SW28, ou equivalente. Sobrenadante densidade sobreposição ajustado com ~ 10 ml de tampão de carbonato de sódio 100 mM gelado (pH 11,5) e 0,5-1 ml de gelado HLM para encher o tubo. </li><li> Centrifugar a 4 ° C a 130,000 g durante 45 min, utilizando rotor basculante SW28. Permitir rotor a costa a uma parada (rotor desaceleração = 0). </li><li> Recolher a camada flutuante, ajustar para 10% de sacarose e transferir para a parte inferior de um tubo de ultracentrífuga 13,2 ml para um rotor SW41Ti, ou equivalente. Sobreposição densidade ajustada sobrenadante com cerca de 5 ml de tampão de carbonato de sódio 100 mM gelado (pH 11,5) e 0,5 mL de HLM gelada. </li><li> Centrifugar a 4 ° C a 274,000 g durante 60 min utilizando SW41Ti rotor basculante. Permitir rotor a costa a uma parada (rotor desaceleração = 0) para minimizar a interrupção da camada de gotículas de lipídios. </li><li> Recolher cuidadosamente a camada superior flutuante (Figura 2D), contendogotículas lipídicas com uma pipeta de 1 ml e repita o passo 3.4. </li><li> Centrifugar a 4 ° C a 274,000 g durante 30 min utilizando SW41Ti rotor basculante. Permitir rotor a costa a uma parada (rotor desaceleração = 0). </li><li> Recolher cuidadosamente a camada flutuante de cor branca top contendo gotículas lipídicas com uma pipeta Pasteur dobrada em três tubos de 1,5 ml contendo 100 mL HLM. </li></ol> Fração gotículas lipídicas Caracterização (Protocolos 1 e 2) A recuperação e a pureza da fracção de gotículas de gordura pode ser verificada por Western Blot combinada com luz ou microscopia electrónica. Além disso, as alíquotas após diferentes passos de centrifugação pode ser recolhida e retida para determinar a eficiência de purificação. Em Western Blotting, é mais apropriado comparar o volume da fração de gotículas de gordura que representa o equivalente a todo o lisado celular de comparar gotículas lipídicas para outras frações de membrana em tEle função do teor de proteína total de 24.

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Chem. 279, 3787-3792 (2004).</li><li>Fujimoto, Y., 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=Identification+of+major+proteins+in+the+lipid+droplet-enriched+fraction+isolated+from+the+human+hepatocyte+cell+line+HuH7.">Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.</a> Biochim. Biophys. Acta. , 47-59 (2004).</li><li>Sato, S., 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=Proteomic+profiling+of+lipid+droplet+proteins+in+hepatoma+cell+lines+expressing+hepatitis+C+virus+core+protein.">Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.</a> J. Biochem. 139, 921-930 (2006).</li><li>Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G., Prohaska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+stomatin+with+lipid+bodies.">Association of stomatin with lipid bodies.</a> J. Biol. Chem. 279, 23699-23709 (2004).</li><li>Zhang, H. N., 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=Proteome+of+skeletal+muscle+lipid+droplet+reveals+association+with+mitochondria+and+apolipoprotein+A-I.">Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.</a> J. Proteome Res. 10, 4757-4768 (2011).</li><li>Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R., McManaman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+reveal+a+link+between+the+endoplasmic+reticulum+and+lipid+secretory+mechanisms+in+mammary+epithelial+cells.">Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.</a> Electrophoresis. 21, 3470-3482 (2000).</li><li>Brasaemle, D. L., Wolins, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+lipid+droplets+from+cells+by+density+gradient+centrifugation.">Isolation of lipid droplets from cells by density gradient centrifugation.</a> Curr. Protoc. Cell Biol. , 3.15.1-3.15.12 (2005).</li><li>Long, A. 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=Lipid+droplet+de+novo+formation+and+fission+are+linked+to+the+cell+cycle+in+fission+yeast.">Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.</a> Traffic. 13, 705-714 (2012).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Cho, S. Y., 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=Identification+of+mouse+Prp19p+as+a+lipid+droplet-associated+protein+and+its+possible+involvement+in+the+biogenesis+of+lipid+droplets.">Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.</a> J. Biol. Chem. 282, 2456-2465 (2007).</li><li>Ding, Y. B., Wu, Y. B., Zeng, R., Liao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+lipid+droplet-associated+proteins+in+primary+adipocytes+of+normal+and+obese+mouse.">Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse.</a> Biochim. Biophys. Acta. 44, 394-406 (2012).</li><li>Jagerstrom, S., 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=Lipid+droplets+interact+with+mitochondria+using+SNAP23.">Lipid droplets interact with mitochondria using SNAP23.</a> Cell Biol. Int. 33, 934-940 (2009).</li><li>Xu, N. Y., 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+FATP1-DGAT2+complex+facilitates+lipid+droplet+expansion+at+the+ER-lipid+droplet+interface.">The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface.</a> J. Cell Biol. 198, 895-911 (2012).</li><li>Ozeki, S., Cheng, J. L., Tauchi-Sato, K., Hatano, N., Taniguchi, H., Fujimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+localizes+to+lipid+droplets+and+induces+their+close+apposition+to+the+endoplasmic+reticulum-derived+membrane.">Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane.</a> J. Cell Sci. 118, 2601-2611 (2005).</li><li>Yang, H. Y., Galea, A., Sytnyk, V., Crossley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+size+of+lipid+droplets:+lipid+and+protein+factors.">Controlling the size of lipid droplets: lipid and protein factors.</a> Curr. Opin. Cell Biol. 24, 509-516 (2012).</li><li>Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., Schneiter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J. Cell Sci. 124, 2424-2437 (2011).</li><li>Pu, J., Ha, C. W., Zhang, S., Jung, J. P., Huh, W. K., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactomic+study+on+interaction+between+lipid+droplets+and+mitochondria.">Interactomic study on interaction between lipid droplets and mitochondria.</a> Protein Cell. 2, 487-496 (2011).</li><li>Shaw, C. S., Jones, D. A., Wagemakers, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+distribution+of+mitochondria+and+lipid+droplets+in+human+muscle+fibres.">Network distribution of mitochondria and lipid droplets in human muscle fibres.</a> Histochem. Cell Biol. 129, 65-72 (2008).</li><li>Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gregarious+lipid+droplet.">The gregarious lipid droplet.</a> J. Biol. Chem. 283, 28005-28009 (2008).</li><li>Brasaemle, D. L., Wolins, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+fat:+an+evolving+model+of+lipid+droplet+assembly+and+expansion.">Packaging of fat: an evolving model of lipid droplet assembly and expansion.</a> J. Biol. Chem. 287, 2273-2279 (2012).</li></ol>

Os autores declaram não haver interesses financeiros concorrentes.

<html> As etapas críticas dentro deste protocolo Certifique-se para ser consistente com os meios e as densidades celulares durante o crescimento das células em cultura. Gotículas lipídicas celulares são únicos na medida em que as suas proteínas associadas são altamente dependentes do ambiente no qual as células são cultivadas 17. Portanto, os meios de comunicação em que as células são cultivadas e da densidade das células devem ser c...

Gotículas lipídicas celulares são organelas dinâmicas que servem múltiplas funções nas células. Eles são centros de armazenamento de lípidos neutros, que podem ser convertidas em energia ou utilizadas para a síntese de fosfolípidos. As gotículas de desempenhar um papel central em condições fisiológicas e patológicas, incluindo a aterosclerose, obesidade e doenças metabólicas relacionadas, e também as doenças infecciosas 1,2. Além disso, eles são fontes interessantes para combustíveis de biodiesel. Muita informação sobre os papéis celulares de gotículas lipídicas foi obtida a partir de análise proteômica e lipidomas de gotículas purificados a partir de organismos de grande alcance 3. Estes organismos incluem as bactérias, levedura 4,5 6-11, plantas 12,13, nematóides 14, e voa 15,16. Dado o interesse no papel de gotículas lipídicas em doenças metabólicas humanos, as gotas foram também isolados a partir de células animais em cultura e umtecidos nimal. Linhas de células cultivadas têm incluído 3T3-L1 adipócitos 17, de ovário de hamster chinês (CHO), células K2 18, 19,20 hepatocyes humanos, e linhas de células epiteliais 21. Tecidos animais a partir do qual as gotas foram isolados incluíram rato músculo esquelético 22, fígado 23, e glândulas mamárias 23. Como mencionado acima, o objetivo da maioria dos estudos de isolamento gota é realizar análise proteômica sobre os fatores ligados e lipidomas no neutro e fosfolipídios. Desde lipídios neutros - os mais numerosos componentes de gotículas lipídicas - são menos densos do que a maioria dos outros materiais celulares, o isolamento de gotículas tem sido tradicionalmente realizada usando gradiente de densidade centrifugação. Essa técnica é a peça central de ambas as preparações apresentadas aqui. As técnicas anteriores são combinados e 6,24 modificado para uma apresentação visual do isolamento de gotículas a partir de células de levedura de fissão cultivadase células humanas noncultured obtido a partir de tecido placentário. O objectivo é o de mostrar a ampla aplicabilidade desta técnica, escolhendo dois tipos de células muito diferentes de pontos para o isolamento a partir das gotículas. Esta técnica deve ser útil para aqueles que desejam isolar gotas da maioria dos organismos. Protocolo 1 descreve o isolamento de gotículas lipídicas a partir da levedura de fissão, Schizosaccharomyces pombe, a qual tem sido utilizada como um modelo para observar a formação de gotículas durante a divisão celular eucariótica 25. A levedura Saccharomyces cerevisiae brotamento tem sido amplamente utilizado como organismo-modelo para estudar a biologia das gotículas de lípidos. Protocolo n ° 1 é aplicável a ambos os organismos e as diferenças nos preparativos são realçados. Protocolo 2 descreve o isolamento de gotículas lipídicas de células das vilosidades da placenta, que são, por sua vez obtida a partir de placenta humana prazo. Ocoleção de placentas prazo proporciona uma oportunidade única de forma segura e eticamente obter 200-250 g de tecido humano prontamente disponível 26, que contém um número significativo de gotículas lipídicas. Isto está em contraste com a maior parte do trabalho de isolamento de gotículas de gordura em eucariotas superiores, onde as gotículas são originários a partir de células cultivadas. Nestes estudos, os ácidos gordos são, muitas vezes adicionado à cultura para promover a síntese de lípidos neutros e, assim, o crescimento de gotículas. Isto está em contraste com o trabalho aqui onde gotículas lipídicas são formadas sob condições nativas no tecido placentário. As purezas das fracções de gotículas de lípido são determinados por análise de Western Blot, utilizando anticorpos de marcadores de organelos. Estes dois protocolos irá produzir fracções de gotículas lipídicas que são adequados para análise proteómica e lipidomas subsequente.

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			<td height="21" style="height:21px;width:353px;">PROTOCOL #1:&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.Growing yeast cells and converting to spheroplasts</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Edinburgh Minimal Media (EMM)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2005</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Yeast extract with 5 supplements (YE5S)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2011</td>
			<td style="width:167px;">YE5S media with 225 mg/ml of each supplement: adeninie, histidine, leucine, lysine, uracil. The equivalent for budding yeast would be YPD.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YPD powder</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">1875</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Lytic Enzyme</td>
			<td>MP Biomedicals</td>
			<td style="width:119px;">215352610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Enzymes from Trichoderma harzianum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">L1412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymolayse-20T</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">N0766391</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td style="width:119px;">D-3922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-544-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic transfer pipette</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">137115AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 liter glass bottle</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml flask</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">2.8 liter flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="20" style="height:20px;">2. Yeast lipid droplet isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll 400</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce Homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiberlite* F15-6x100y Fixed-Angle Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003698</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROTOCOL #2&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1. Placental villous cells isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable underpads</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">23666062</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Autoclavable pan (container), 3L</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">1336110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors, sharp-sharp, straight</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1406011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">London Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1108002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7b Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1127020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor blades</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S65921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screen cup for CD-1</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S1145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 mesh screen&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S0770</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand cell stainers 100&#956;m</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">22363549</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 mm Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">NC9054771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S642</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P386</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S374</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">D16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.5% trypsin 10x</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15090046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I grade II, from bovine pancreas</td>
			<td>Roche Applied Science</td>
			<td style="width:119px;">10104159001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate solution</td>
			<td>Sigma-aldrich</td>
			<td style="width:119px;">S8761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">500 ml Erlenmeyer flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">250 ml beakers</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">10 ml serological pipettes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">DMEM</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Lipid droplets isolation from villous placental cells</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Sucrose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Carbonate&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Clear centrifuge tubes 14x89mm (for SW41)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable borosilicate glass pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Biological safety hood&#160;</td>
			<td style="width:163px;">Thermo-Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Western blot</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye 680 Goat Anti-Rabbit IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-68071</td>
			<td>dilution 1:15000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye&#160; 800CW Goat Anti-Mouse IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-32210</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">NuPAGE&#174; Novex&#174; 12% Bis-Tris gels</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">NP0341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #1</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Erg6p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Dpm1p</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113686</td>
			<td>4 &#956;g/ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Por1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Pma1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Vma1p (anti-ATP6V1A)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113745</td>
			<td>0.5 &#956;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;"></td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #2</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">perilipin 2 (anti-ADFP)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab52355</td>
			<td>2 &#956;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">calnexin</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">2679</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">GM130</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb40533</td>
			<td>dilution 1:25</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">COX IV</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">4850</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MEK1</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb38775</td>
			<td>dilution 1:50</td>
		</tr>
	</tbody>
</table>

isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas

Gotículas lipídicas são organelas dinâmicas que podem ser encontrados na maioria das células procariotas e eucariotas certos. Estruturalmente, as gotículas constituídas por um núcleo de lípidos neutros rodeadas por uma monocamada de fosfolipido. Uma das técnicas mais úteis na determinação dos papéis celulares de gotículas foi identificação proteoma de proteínas ligadas, que podem ser isolados, juntamente com as gotas. Aqui, dois métodos são descritos para isolar gotículas lipídicas e suas proteínas ligadas a partir de duas amplas eucariontes: levedura de fissão e as células das vilosidades da placenta humana. Apesar de ambas as técnicas têm diferenças, o método main - gradiente de densidade centrifugação - é compartilhado por ambas as preparações. Isto demonstra a ampla aplicabilidade das técnicas de isolamento de gotículas apresentados. No primeiro protocolo, as células de levedura são convertidas em esferoplastos por digestão enzimática das paredes celulares. Os esferoplastos resultantes são então gently lisadas em um homogeneizador folgadas. Ficoll é adicionado ao lisado para providenciar um gradiente de densidade, e a mistura é centrifugada três vezes. Depois da primeira rotação, as gotículas lipídicas são localizadas na camada flutuante de cor branca dos tubos de centrífuga, juntamente com o retículo endoplasmático (ER), a membrana de plasma, e vacúolos. Dois rotações subsequentes são utilizados para remover estes três organelos. O resultado é uma camada que tem apenas gotículas e as proteínas ligadas. No segundo protocolo, as células das vilosidades da placenta são isolados a partir de placenta humana prazo por digestão enzimática com tripsina e DNase I. As células são homogeneizadas num homogeneizador folgada. De baixa velocidade de centrifugação e de velocidade média passos são usadas para remover as células não quebradas, os detritos celulares, os núcleos, e as mitocôndrias. É adicionada sacarose ao homogenato para providenciar um gradiente de densidade, e a mistura é centrifugada para separar as gotículas lipídicas a partir do outro cellulafracções r. A pureza das gotículas lipídicas em ambos os protocolos, é confirmada por análise Western Blot. As frações de gotículas de ambas as preparações são adequados para a análise proteômica e lipidomas subseqüente.

Se a centrifugação em gradiente de densidade funcionou como esperado, a camada flutuante deve conter gotículas lipídicas e ser desprovido de outros organelos toda a progressão dos spins de alta velocidade. Para Protocolo 1, Western Blot foram realizadas com anticorpos para marcadores gotículas lipídicas (Erg6p) e organelas que foram encontrados para interagir com gotículas lipídicas em levedura, ER (Dpm1p), mitocôndria (Por1p), membrana plasmática (Pma1p), e vacúolos (Vma1p). </p...

Watch this Scientific Journal Video about Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas at JoVE.com

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Duas técnicas de isolamento de gotículas lipídicas celulares a partir de 1) células de levedura e 2) placentas humanas estão apresentadas. A peça central de ambos os processos é a centrifugação de gradiente de densidade, onde a camada flutuante resultante contendo as gotículas podem ser facilmente visualizadas por olho, extraiu-se, e quantificada por análise de Western Blot quanto à pureza.

Isolamento de celulares lipídicas Gotas: Duas técnicas de purificação Começando a partir de células de levedura e placentas humanas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

isolation-cellular-lipid-droplets-two-purification-techniques

Research

JoVE Journal

Bioengineering

19.5K visualizações.  University of Tennessee.  Duas técnicas de isolamento de gotículas lipídicas celulares a partir de 1) células de levedura e 2) placentas humanas estão apresentadas. A peça central de ambos os processos é a centrifugação de gradiente de densidade, onde a camada flutuante resultante contendo as gotículas podem ser facilmente visualizadas por olho, extraiu-se, e quantificada por análise de Western Blot quanto à pureza. 

Vídeo: Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Bioengenharia

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Desenvolvimento, expansão e<em&gt; In vivo</em&gt; Monitorização das células NK humanas de células estaminais embrionárias humanas (hESCs) e e induziu células-tronco pluripotentes (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lipid Bilayer Vesicle Geração Usando Microfluidic jateamento

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento de células do cólon tumor primário Humanos a partir de tecidos cirúrgicos e cultivando-as Diretamente macias elásticas Substratos para Traction Citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Técnicas para a Evolução da levedura Pentose-fermentação robusta para bioconversão de transformação de lignocelulose em etanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Captação de novas nanopartículas revestidos de lipídeos contendo Falcarindiol por células-tronco mesenquimais humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolamento de fibroblastos dérmicos humanos adultos da pele abdominal e geração de células-tronco pluripotentes induzidas usando um método não integrador

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medição potencial de ação óptica unicelular em cardiomiócitos derivados de células-tronco pluripotentes induzidos por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...