K. Wong, Wong STC são suportados pelo financiamento do Funcional e Molecular Imaging Center, Brigham e hospital das mulheres K. Wong, Wong STC são suportados pelo Centro de Biotecnologia e Informática, The Methodist Hospital Research Institute, Weill Cornell Medical College. KK Kwong, I. Chen, JQ Ren são suportados pelo financiamento do A. Athinoula Martinos Center for Biomedical Imaging, Massachusetts General Hospital. Lenuta Kloetzer, Braden Kuo são suportados por fundos do Centro de Pesquisa neuroentérico, Massachusetts General Hospital Os autores agradecem os drs. Q. Xu X. Zeng e de imagem Optical Lab, Brigham e do Hospital da Mulher para ajuda técnica.

<ol><li> Feminino HO-1-luciferase camundongos transgênicos (idade de 4-6 semanas) pode ser comprado de Taconic Farms, Inc. Após a chegada, os ratos são colocados em uma instalação de alojamento dos animais durante pelo menos 4 dias para permitir acomodação. </li><li> Preparação para a imagem de bioluminescência: <ol style="list-style-type:lower-alpha;"><li> Pesar cada rato HO-1. Peso do animal é necessário para calcular a dosagem de luciferina. </li><li> Depilação: Use um cotonete para aplicar removedor de pêlos Nair ® sobre o abdome e / ou traseira do animal. À espera de 5 a 10 segundos. Utilize um cotonete limpo para limpar o cabelo. Mergulhe um pedaço de tecido em água destilada para limpar o cabelo restante limpo. </li><li> Coloque um papel não-fluorescente preto (Strathmore Artagain ® papel preto) na plataforma de imagem de uma estação de 100 IVIS para reduzir o ruído de fundo. </li><li> Prepare luciferina (de Xenogen Corporation) solução na concentração de 7,5 mg / ml (dissolvida em estéril H 2 O). A dose de luciferina é o peso corporal 65,5 mg / kg. Assim, um mouse de 20 gramas requer injeção intraperitoneal de solução de luciferina 0,175 ml. Para conseguir isso, um volume de injeção única de luciferina solução (0.175ml) é pré-carregado a uma seringa com uma agulha de calibre 26 </li></ol></li><li> Gua Sha procedimento: Gua Sha é aplicado apenas uma vez antes de executar o protocolo de imagem de bioluminescência. Desde Gua Sha não é doloroso, o rato só precisa ser brevemente anestesiados com isoflurano para manter a calma. Gua Sha processo inclui as seguintes etapas: <ol style="list-style-type:lower-alpha;"><li> Aplicar óleo de cozinha ou água destilada para lubrificar áreas da pele alvo de Gua Sha algumas vezes durante o procedimento de Sha Gua. </li><li> Repetidamente raspar o cabelo região livre da parte traseira do mouse em vigor suave, mas firme com uma colher de sopa de cerâmica ou uma colher de plástico. </li><li> A raspagem continua até que a pele fica vermelha de volta, que é um sinal de extravasamento de sangue por via subcutânea, geralmente atingida dentro de 2 a 3 minutos. </li></ol></li><li> Imagem de bioluminescência pode ser iniciado imediatamente ou várias horas após Gua Sha como HO-1 upregulation é construída lentamente ao longo de algumas horas. O procedimento para a imagem latente da bioluminescência é: <ol style="list-style-type:lower-alpha;"><li> Anestesiar o rato em uma câmara de indução da anestesia preenchido com uma mistura de ar grade isoflurano (1,5%) e médicos. </li><li> Uma vez que o mouse está anestesiado, mova o mouse para a câmara de imagem na estação de imagem óptico IVIS 100. Posicione o mouse em decúbito dorsal (barriga para cima). A câmara de imagem é continuamente infundidos com 1,5% de isoflurano. A plataforma de imagem é aquecida a 37 ° C para manter o mouse quente. </li><li> Definir a aquisição de imagens em &quot;binning médio&quot; e tempo de exposição definido para ser 30 segundos. Começar a adquirir imagens. Configurar a máquina para repetir a aquisição de imagem a cada três minutos, manual ou automaticamente. Após a aquisição da primeira imagem, uma região de interesse (ROI) é desenhada para cobrir o tórax, abdominal, e região da cabeça. Este ROI é então copiado e colado às imagens a seguir usando &quot;Living Imagem ®&quot;, software que acompanha o IVIS 100 estação. </li><li> Intensidade do sinal é medido em fótons por segundo. Tempo para marcar a intensidade do sinal para chegar ao seu valor de pico e manter a imagem o mouse por cerca de 5 a 10 minutos após o horário de pico. </li><li> Quando a aquisição de imagens na posição supina é feito, flip o mouse e colocá-lo na posição prona (back up). Continue imagem do animal de 5 a 10 minutos com o ROI agora desenhadas para cobrir as costas ea região da cabeça no uso do &quot;imagem viva ®&quot; software. A escolha da imagem na posição prona, após a posição supina é arbitrária. Pode-se optar por começar com a posição prona, dependendo de os principais órgãos de interesse. Outra opção é a imagem posições supina e propenso a experimentos separados. </li><li> Quando a aquisição de imagem na posição prona é terminado, desligue o isoflurano eo movimento do mouse da câmara de imagem para a câmara de indução para se recuperar. A câmara de indução é agora infundido com ar médica e só o mouse normalmente acorda em menos de um minuto. </li><li> Salvar dados de imagem para pós-processamento. </li><li> Após o estudo de imagem inicial, aquisições de imagem adicionais são realizadas por vários dias após Gua Sha. Uma agenda de amostra é repetida imagem na hora 12 ª, 24 ª hora, 36 ª hora, 48 ª hora, 72 horas nd e 120 ª hora, mas o momento exato do calendário de imagem é flexível. Sessões de imagem adicionais podem ser adicionados se um longo acompanhamento é necessário. Antes de imagem, repita o procedimento de remoção do cabelo se o cabelo tem crescido de volta. </li></ol></li><li> Para o grupo controle, repita todo o procedimento, mas ignorar Gua Sha (passo 3). Se o mesmo grupo de ratos é utilizado tanto para controle e Gua Sha, o controle de experiment deve ser realizada preferencialmente antes do procedimento sha Gua ou, pelo menos, um mês depois. </li></ol> Resultados representativos: As imagens mostram a bioluminescência na figura 1 em upregulation vivo da HO-1 em resposta a Gua Sha. O gráfico da Figura 2 mostra a mudança quantitativa temporal sobre 120 horas em fluxo óptico (fótons / seg) de todo o corpo do mouse mesmo relacionadas com Gua Sha ... <img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="figura 1" border="0" /> Figura 1. Da esquerda para a direita, as imagens representativas da vista frontal (supino) do mouse mesmo antes de Gua Sha, às 18 horas, 36 horas e 120 horas após a Gua Sha, respectivamente. Depois de Gua Sha, observa-se o progresso do aumento significativo intensidade de sinal em múltiplos órgãos, que abrangem as regiões do trato gastrointestinal, o trato genital, o fígado, rins (do ponto de vista de volta, não mostrado), e outros. Por favor, <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">clique aqui</a> para uma versão ampliada da figura 1. <img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="figura 2" border="0" /> Figura 2. Mudança quantitativa do fluxo (fótons / seg) de todo o corpo rastreadas mais de 120 horas após Gua Sha no mouse mesmo da Figura 1. Por favor, <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">clique aqui</a> para ver uma versão ampliada da figura 2.

<ol>
	<li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+potential+therapeutics+based+on+in+vivo+spatiotemporal+transcription+patterns+of+heme+oxygenase-1.">Selection of potential therapeutics based on in vivo spatiotemporal transcription patterns of heme oxygenase-1.</a> J Mol Med. 80, 655-6564 (2002).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+functional+analysis+of+transgenes+in+mice+using+whole+body+imaging+of+luciferase+expression.">Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression.</a> Transgenic Res. 10, 423-434 (2001).</li><li>Contag, C. H., Stevenson, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+patterns+of+heme+oxygenase-1+transcription.">In vivo patterns of heme oxygenase-1 transcription.</a> J Perinatol. 21, 119-1124 (2001).</li><li>Nielsen, A., Knoblauch, N. T., Dobos, G. J., Michalsen, A., Kaptchuk, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Gua+Sha+treatment+on+the+microcirculation+of+surface+tissue:+a+pilot+study+in+healthy+subjects.">The effect of Gua Sha treatment on the microcirculation of surface tissue: a pilot study in healthy subjects.</a> Explore (NY). 3, 456-466 (2007).</li><li>Schwickert, M. E., Saha, F. J., Braun, M., Dobos, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gua+Sha+for+migraine+in+inpatient+withdrawal+therapy+of+headache+due+to+medication+overuse.">Gua Sha for migraine in inpatient withdrawal therapy of headache due to medication overuse.</a> Forsch Komplementmed. 14, 297-300 (2007).</li><li>Tsai, P. S., Lee, P. H., Wang, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographics,+training,+and+practice+patterns+of+practitioners+of+folk+medicine+in+Taiwan:+a+survey+of+the+Taipei+metropolitan+area.">Demographics, training, and practice patterns of practitioners of folk medicine in Taiwan: a survey of the Taipei metropolitan area.</a> J Altern Complement Med. 14, 1243-1248 (2008).</li><li>Nielsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gua+sha+research+and+the+language+of+integrative+medicine.">Gua sha research and the language of integrative medicine.</a> J Bodyw Mov Ther. 13, 63-72 (2009).</li><li>Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+gene+expression+in+living+mammals+using+a+bioluminescent+reporter.">Visualizing gene expression in living mammals using a bioluminescent reporter.</a> Photochem Photobiol. 66, 523-531 (1997).</li><li>Cui, K., Xu, X., Zhao, H., Wong, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+mediates+transcriptional+activation+of+the+heme+oxygenase-1+gene+in+response+to+hypoxia.">Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.</a> J Biol Chem. 272, 5375-5381 (1997).</li><li>Maines, 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+heme+oxygenase+system:+a+regulator+of+second+messenger+gases.">The heme oxygenase system: a regulator of second messenger gases.</a> Annu Rev Pharmacol Toxicol. 37, 517-554 (1997).</li><li>Wunder, C., Potter, R. 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The authors have nothing to disclose.

Transcrição de HO-1, uma forma induzível da heme oxigenase, é regulada por vários fatores, incluindo heme, peróxido de hidrogênio, radiação UV, hipóxia e stress físico. Imagens de corpo inteiro, como o relatado HO-1 protocolo de bioluminescência fornece uma visão rápida de expressão de HO-1 sistêmica em múltiplos órgãos. Em pequenos animais, imagem de bioluminescência permite alta sensibilidade in vivo em tempo real avaliação quantitativa longitudinal óptico de alterações na expressão genética...

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<td>Luciferin</td>
<td>&nbsp;</td>
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<td>IVIS 100</td>
<td>&nbsp;</td>
<td>Caliper </td>
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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

Gua Sha é uma terapia popular tradicional chinesa que emprega raspagem da pele para causar subcutânea extravasamento de sangue microvascular e contusões. O protocolo de imagem óptica bioluminescente da HO-1-<em&gt; Luciferase</em&gt; Camundongos transgênicos reportados neste manuscrito oferece uma rápida<em&gt; In vivo</em&gt; Ensaio da upregulation da heme oxigenase-1 (HO-1) a expressão gênica em resposta ao procedimento Sha Gua. HO-1 tem sido conhecido para fornecer citoproteção contra o estresse oxidativo. A regulação alta de HO-1, avaliados pela saída bioluminescência, é pensado para representar uma resposta antioxidante de produtos hemoglobina circulante lançado pela Gua Sha. Gua Sha foi administrada por golpes repetidos de uma borda colher suave sobre a pele lubrificada nas costas ou outra parte do corpo alvo do camundongo transgênico até petéquias (hemorragias lasca) ou equimoses (manchas roxas) indicativo de extravasamento de sangue de capilares subcutâneos foi observada. Depois de Gua Sha, sessões de imagem de bioluminescência foram realizadas diariamente durante vários dias para acompanhar a dinâmica de expressão de HO-1 em vários órgãos internos.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha, raspagem de pele tradicional chinesa terapêutica, causa extravasamento de sangue por via subcutânea microvascular. Relatamos um protocolo de imagem de bioluminescência de HO-1-<em&gt; Luciferase</em&gt; Camundongos transgênicos para demonstrar que Gua Sha upregulates heme oxigenase-1 (HO-1) em vários órgãos.

Imagem de bioluminescência de heme oxigenase-1 Upregulation no Procedimento de Gua Sha

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

14.0K visualizações.  Massachusetts General Hospital, Harvard Medical School. Gua Sha, raspagem de pele tradicional chinesa terapêutica, causa extravasamento de sangue por via subcutânea microvascular. Relatamos um protocolo de imagem de bioluminescência de HO-1- Luciferase Camundongos transgênicos para demonstrar que Gua Sha upregulates heme oxigenase-1 (HO-1) em vários órgãos.

Vídeo: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Um procedimento de cirurgia Craniotomia para Brain Imaging crônica

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Biologia

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro e in vivo de imagens Gene Reporter bioluminescência de células estaminais embrionárias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murino Procedimento de Transplante Renal

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagem Funcional de gordura marrom em ratos com FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Monitorização das células-autônomas Ritmos relógio circadiano de expressão genética dos Repórteres Bioluminescência luciferase

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Investigando interações proteína-proteína em células vivas usando a transferência de Bioluminescência Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Imagem de peróxido mitocondrial em células vivas de levedura usando mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Autor em destaque: Avançando na pesquisa mitocondrial - biossensor mtHyper7 para análise subcelular 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Imagem de mtHyPer7, um biossensor ratiométrico para peróxido mitocondrial, em células vivas de levedura

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...