Somos gratos aos Drs. Aravinthan Samuel e Marc Gershow que ajudaram a desenvolver a versão inicial do aparelho comportamental e Matthew Batie que modificou o aparelho comportamental. Esta pesquisa foi apoiada pelo Trustee Grant do Hospital Infantil de Cincinnati, JST/PRESTO, March of Dimes e NIH R01 GM107582 a F.N.H.

1. Preparação de Moscas
<ol><li>Experimentos de Luz Escura (LD)
	<ol>
<li>Levante moscas em incubadoras (25 °C/40-60% umidade relativa (RH)) sob ciclos leves de 12 horas/escuros de 12 horas (LD). A intensidade de luz das incubadoras é de ~500-1.000 lux.</li>
		<li>Duas incubadoras são necessárias para completar os ensaios comportamentais durante um período de 24 horas. Ambas as incubadoras devem ter uma luz programável com funções ON OFF. Eles também devem ter portas sólidas que não são permeáveis à luz (ou seja, sem vidro ou plexiglass). Nota: Uma incubadora deve ser designada como incubadora "day" e definida para um ciclo LD de 12 horas de luz e 12 horas escuras. A segunda incubadora deve ser designada como incubadora "noturna" e definida para o inverso do primeiro com 12 horas de ano, seguida de 12 horas de luz. A incubadora noturna deve ser colocada no quarto acessível no escuro, de tal forma que moscas que experimentam condições noturnas podem ser facilmente acessadas para os experimentos.</li>
		<li>Coloque os frascos de mosca em incubadoras dia ou noite. Colete moscas recém-eclodidas em um frasco fresco, 20-30 por ensaio e mantenha na mesma incubadora por 2-3 dias.</li>
		<li>Após 2-3 dias, use moscas para o ensaio comportamental de preferência de temperatura.<ol>
<li>Para experimentos diurnos (hora de Zeitgeber (ZT) 0-12), colete moscas da incubadora do dia.</li>
			<li>Logo antes dos experimentos comportamentais, tire os frascos de mosca coletados da incubadora do dia.</li>
			<li>Para experimentos noturnos (ZT 13-24), colete moscas da incubadora noturna.</li>
			<li>Logo antes dos experimentos comportamentais, tire os frascos de mosca coletados da incubadora noturna, enrole com papel alumínio e coloque em uma caixa na sala escura sob uma lâmpada vermelha. Nota: Uma vez que o ensaio comportamental de preferência de temperatura é realizado sob a escuridão para os experimentos noturnos, a exposição à luz às moscas deve ser evitada até o final dos experimentos comportamentais. Nota: As moscas não devem ser expostas ao dióxido de carbono no dia em que os experimentos ocorrerão.</li>
		</ol>
</li>
	</ol></li>
	<li>Experimentos de Escuridão Constante (DD)
	<ol>
<li>
Dia DD
		<ol>
<li>Uma incubadora extra é necessária para experimentos do dia DD, que chamamos de incubadora de "transição" para o resto do manuscrito. A incubadora de transição deve ser colocada na sala acessível no escuro, de tal forma que moscas que experimentam condições dD podem ser facilmente acessadas para os experimentos. Um exemplo de cronograma de luz para uma incubadora de transição teria a luz ACESA das 13:00 às 19:00 e a luz OFF às 19:00 às 13:00(Figura 1).</li>
			<li>Recolhe as moscas que foram levantadas na incubadora do dia. Coloque os frascos de mosca na incubadora de transição entre 13h e 19h, quando a luz estiver acesa. Desta forma, as moscas ficam devidamente expostas à luz até às 19h, momento em que a luz se desliga.</li>
			<li>No dia seguinte, antes das 13h, em condições escuras, tire os frascos de mosca da incubadora de transição, enrole-os com papel alumínio e coloque-os em uma caixa. Mantenha a caixa em qualquer incubadora por mais um dia.</li>
		</ol>
</li>
		<li>
Noite DD
		<ol>
<li>Colete as moscas que foram levantadas na incubadora noturna no escuro sob uma lâmpada vermelha. Ou, adicionalmente, coletar as moscas quando a luz da incubadora noturna estiver acesa.</li>
			<li>Enrole os frascos coletados com papel alumínio no escuro sempre que as luzes estiverem desligadas e coloque os frascos em uma caixa. Mantenha a caixa em qualquer incubadora por mais dois dias(Figura 1B). Nota: Uma vez que o ensaio comportamental de preferência de temperatura é realizado sob a escuridão para os experimentos noturnos, a exposição à luz às moscas deve ser evitada até o final dos experimentos comportamentais.</li>
		</ol>
</li>
	</ol></li>
	<li>Experimentos de Luz Constante (LL)
	<ol>
<li>
Dia LL
		<ol>
<li>Uma incubadora extra é necessária para experimentos diários ll. Esta incubadora mantém a condição LL (25 °C, 800 lux), com a luz ACESA continuamente.</li>
			<li>Recolher moscas que foram levantadas na incubadora do dia. Coloque os frascos de mosca na incubadora LL a qualquer momento durante seu "dia".</li>
		</ol>
</li>
		<li>
Noite LL
		<ol>
<li>Recolher moscas que foram criadas na incubadora noturna. Transfira os frascos de mosca da incubadora noturna para a incubadora LL durante o tempo em que a luz na incubadora noturna estiver acesa. Nota: Por exemplo, a luz se apaga às 7h da manhã na incubadora noturna. Transfira os frascos de mosca da incubadora noturna para a incubadora LL antes das 7h e mantenha os frascos na incubadora LL por mais 4 dias. Nota: No dia 4 em condições de LL, a oscilação da atividade locomotor é abolida15,16, enquanto o TPR ainda é sustentado 3.</li>
		</ol>
</li>
	</ol></li>
</ol>2. O aparelho para o ensaio comportamental de preferência de temperatura
<ol><li>Coloque uma tampa de plexiglass (29 cm x 19,2 cm)(Figura 4) em uma placa de alumínio.</li>
	<li>Monitore a temperatura do ar entre a placa e a tampa. Seis sondas de temperatura são anexadas em várias posições no interior da tampa dentro de uma das pistas(Figura 2). Nota: Certifique-se de que as sondas não toquem na placa de alumínio ou na tampa do plexiglass. A temperatura do ar deve ser definida para um gradiente de 18-32 °C.</li>
	<li>Coloque o aparelho em uma sala ambiental mantida em 25 °C/65-75% RH. Esta sala precisa ser selada de qualquer luz externa. Uma sala ambiental é normalmente equipada com ventiladores para manter uma certa temperatura e umidade. Nota: O ar do ventilador provavelmente perturba um gradiente de temperatura estável no aparelho. Para evitar isso, utilizamos uma folha transparente que cobre a área ao redor do aparelho.</li>
	<li>Prepare um termômetro e higrômetro para verificar a temperatura e a umidade na sala ambiental. Nota: A luz influencia a preferência de temperatura de Drosophila3. A mesma intensidade de luz deve ser fornecida no aparelho uniformemente. A intensidade das luzes da nossa sala ambiental é de ~800 lux. Nota: Quando os experimentos comportamentais forem feitos, coloque um tubo conectado do tanque de CO2 ou forneça perto do orifício da parte superior do aparelho, a fim de anestesiar e se livrar das moscas.</li>
</ol>3. Preparação de aparelhos para uso
<ol><li>Ligue o aparelho por pelo menos 30 minutos para estabelecer o gradiente de temperatura corretamente na superfície da placa(Figura 3).</li>
	<li>Cubra o aparelho comportamental com repelente de água para evitar que as moscas subam as paredes ou o teto da tampa. Limpe o repelente de água em excesso e deixe a tampa por 25-30 minutos para secar.</li>
	<li>Remova qualquer condensação na placa de alumínio. Coloque a tampa do plexiglass na placa de alumínio e fixe com seis grampos C(Figura 2). Nota: É muito importante que a tampa esteja bem selada, se necessário, pode ser usada fita adesiva dupla.</li>
	<li>Deixe a capa por pelo menos 15 minutos. O gradiente da temperatura do ar entre a placa de alumínio e a tampa é criado de 18-32 °C.</li>
</ol>4. Ensaio de comportamento de preferência de temperatura
<ol><li>Carregue as moscas no espaço entre a placa de alumínio e a tampa do plexiglass do aparelho através de pequenos orifícios no centro de cada faixa da tampa (Figuras 2 e 4). Cubra os orifícios com deslizamentos de cobertura para evitar que as moscas escapem.</li>
	<li>Para as condições escuras, desligue todas as luzes da sala ambiental. Uma lâmpada vermelha pode ser usada quando as moscas são colocadas no aparelho. Certifique-se de que as moscas não estão expostas a nenhuma luz, exceto uma lâmpada vermelha até que os experimentos comportamentais estejam prontos.</li>
	<li>Para cada teste, use 20-25 moscas adultas, que não devem ser reutilizadas em testes subsequentes. O ensaio comportamental é realizado por 30 minutos. Tire algumas fotos com ou sem um flash. Tenha cuidado para não fazer muito barulho ou qualquer movimento repentino durante os experimentos.</li>
	<li>Regissuas temperaturas de todas as seis sondas no aparelho. Tome nota da temperatura ambiente, bem como da umidade.</li>
	<li>Anestesiar as moscas do aparelho com gás carbônico, soltar os grampos, remover a tampa do plexiglass e remover as moscas da placa. Após cada experimento, as moscas são descartadas. Limpe qualquer condensação ou umidade da placa. Substitua a tampa da placa e aperte com os grampos em preparação para o próximo experimento.</li>
	<li>Para ter uma representação da preferência de temperatura durante todo o dia, o período de 24 horas é dividido em oito fusos horários, quatro durante o dia e quatro à noite. Por exemplo, usamos estes ZT ou CT 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21 e 22-24. Nota: Uma vez que as variações do fenótipo causadas pelos efeitos de mascaramento são esperadas logo após a luz ser acesa (ZT0) ou OFF (ZT12), não examinamos o comportamento de preferência de temperatura durante esses tempos (ZT ou CT 0-1, 11.5-13 e 23,5-24). Pelo menos cinco ensaios devem ser feitos em cada fuso horário para que os resultados sejam estatisticamente sólidos.</li>
</ol>5. Análise de dados
<ol><li>Calcule o gradiente de temperatura da seguinte forma: Determine onde as sondas de temperatura são colocadas com base nas duas réguas que são colocadas na parte superior e inferior da tampa do plexiglass ao longo das bordas(Figura 2A).</li>
	<li>Estima-se que o gradiente de temperatura entre as sondas de temperatura seja linear. Com base nos locais das sondas de temperatura, bem como em suas temperaturas registradas correspondentes, desenhe linhas representando cada grau de temperatura na posição apropriada nas imagens. Conte o número de moscas localizadas em cada intervalo de grau. Exclua as moscas nas paredes ou no teto da tampa.</li>
	<li>Calcule a porcentagem das moscas em cada faixa de temperatura de cada pista. Uma temperatura preferencial média é calculada somando os produtos da porcentagem de moscas e temperatura de cada intervalo, como mostrado abaixo: % das moscas x 18,5 °C + % das moscas x 19,5 °C + % das moscas x 20,5 °C ....+ % das moscas x 31,5 °C + % das moscas x 32,5 °C.</li>
	<li>Calcule a temperatura preferencial média em cada fuso horário: O comportamento de preferência de temperatura é realizado >5 vezes durante cada fuso horário (ZT 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21 e 22-24). Para calcular a temperatura média preferencial em cada fuso horário, a temperatura média preferencial de cada ensaio é média em conjunto. As barras de erro s.e.m. são iguais ao erro entre as tentativas.</li>
</ol>

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Aqui, ilustramos os detalhes do aparato comportamental de preferência de temperatura e análise do comportamento do TPR. Drosophila exibe as características salientes, robustas e reprodutíveis do TPR controlado pelo relógio. No entanto, nossos dados sugerem que pelo menos dois fatores, luz ambiente e idade, perturbam significativamente os fenótipos comportamentais do TPR.
Observamos que a luz afeta significativamente a preferência de temperatura em Drosophila. É consis...

A temperatura é uma sugestão ambiental onipresente. Os animais apresentam uma variedade de comportamentos para evitar temperaturas prejudiciais e buscar os confortáveis. Drosophila apresentam um comportamento robusto de preferência de temperatura6,7. Quando as moscas são liberadas em um gradiente de temperatura de 18-32 °C, as moscas evitam temperaturas quentes e frias e finalmente escolhem uma temperatura preferencial de 25 °C pela manhã3. Os sensores de temperatura quente são um conjunto de neurônios termossensoriais, neurônios AC, que expressam o potencial do receptor transitório Drosophila (TPR), TRPA16,9. Os sensores de temperatura fria estão localizados nos 3º segmentos de antenas, uma vez que a ablação dos 3º segmentos de antenas causa a falta de evasão de temperatura fria6. Recentemente, foi identificada a proteína TRPP Brivido (Brv)10. Como o Brv é expresso nos 3º segmentos de antenas e media a detecção a frio, o Brv é uma possível molécula de sensoriamento a frio, que é fundamental para o comportamento de preferência de temperatura. Em suma, as moscas usam esses dois sensores de temperatura para evitar as temperaturas quentes e frias e encontrar uma temperatura preferida.
Enquanto os mamíferos geram calor para regular sua temperatura corporal, os ectotherms geralmente adaptam suas temperaturas corporais à temperatura ambiente11. Alguns ectotherms são conhecidos por exibir um comportamento diário de TPR que acredita-se ser uma estratégia para os ectotherms para regular seu BTR12. Para determinar se as moscas apresentavam TPR, repetimos a análise comportamental da preferência de temperatura em vários pontos durante um período de 24 horas. Descobrimos que a Drosophila exibe um TPR diário, que é baixo pela manhã e alto à noite e segue um padrão semelhante ao do BTR em humanos13.
Em Drosophila,há ~150 neurônios de relógio no cérebro. Os neurônios do relógio que regulam a atividade locomotor são chamados osciladores M e E. No entanto, curiosamente, os osciladores M e E não regulam tpr, em vez disso, mostramos que os neurônios do relógio DN2 no cérebro regulam a atividade TPR, mas não locomotor. Esses dados indicam que o TPR é regulado independentemente da atividade locomotor. Notavelmente, o BTR mamífero também é regulado independentemente da atividade locomotor. Estudos de ablação em ratos mostram que o BTR é controlado através de neurônios SCN específicos que visam um subconjunto diferente de neurônios de zona subparaventricular do que aqueles que controlam a atividade locomotor14. Portanto, nossos dados consideram a possibilidade de que o MPI mamífero e a mosca TPR sejam evolucionariamente conservados3, uma vez que tanto a mosca TPR quanto a MMH de mamíferos exibem ritmos de temperatura circadianos dependentes do relógio, que são regulados independentemente da atividade locomotor.
Aqui, descrevemos os detalhes de como analisar o ensaio comportamental do TPR em Drosophila. Este método permite a investigação não apenas do mecanismo molecular e circuitos neurais do TPR, mas também de como o cérebro integra diferentes pistas ambientais e relógios biológicos internos.

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			<td height="41" style="height:41px;width:172px;">Bright Lab Jr. Safelight</td>
			<td style="width:109px;">Amazon</td>
			<td style="width:153px;">#B00013J8UY</td>
			<td style="width:144px;">Red light for dark rooms</td>
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			<td height="61" style="height:61px;width:172px;">Rain X</td>
			<td style="width:109px;">SOPUS products</td>
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			<td style="width:144px;">Water repellent: Apply the plexiglass cover</td>
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			<td height="21" style="height:21px;width:172px;">C-Clamp</td>
			<td style="width:109px;">Home Depot</td>
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			<td height="32" style="height:32px;width:172px;">Temperature/hygrometer</td>
			<td style="width:109px;">Fisher</td>
			<td style="width:153px;">15-077-963</td>
			<td style="width:144px;"></td>
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			<td height="21" style="height:21px;width:172px;"></td>
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			<td height="44" style="height:44px;width:172px;">Peltier devices</td>
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design and analysis of temperature preference behavior and its circadian rhythm in drosophila

O relógio circadiano regula muitos aspectos da vida, incluindo sono, atividade locomotor e temperatura corporal (BTR) ritmos1,2. Recentemente identificamos uma nova produção circadiana Drosophila, chamada ritmo de preferência de temperatura (TPR), em que a temperatura preferencial em moscas sobe durante o dia e cai durante a noite 3. Surpreendentemente, a atividade TPR e locomotor são controladas através de neurônios circadianos distintos3. A atividade locomotor de Drosophila é uma produção comportamental circadiana bem conhecida e tem proporcionado fortes contribuições para a descoberta de muitos genes e mecanismos de relógio circadianos conservados4. Portanto, a compreensão do TPR levará à identificação de mecanismos circadianos moleculares e celulares até então desconhecidos. Aqui, descrevemos como executar e analisar o ensaio TPR. Essa técnica não só permite dissecar os mecanismos moleculares e neurais do TPR, mas também fornece novas percepções sobre os mecanismos fundamentais das funções cerebrais que integram diferentes sinais ambientais e regulam comportamentos animais. Além disso, nossos dados publicados recentemente sugerem que a mosca TPR compartilha características com o mamífero BTR3. Drosophila são ectotherms, em que a temperatura corporal é tipicamente regulada comportamentalmente. Portanto, o TPR é uma estratégia usada para gerar uma temperatura corporal rítmica nessas moscas5-8. Acreditamos que uma exploração adicional do Drosophila TPR facilitará a caracterização dos mecanismos subjacentes ao controle da temperatura corporal nos animais.

Um exemplo do ritmo de preferência de temperatura é mostrado na Figura 5. Se o procedimento de comportamento for feito com sucesso, as moscas devem exibir um TPR no qual preferem uma temperatura baixa pela manhã e temperatura mais alta à noite. O aumento ~1-1,5 °C durante o dia na preferência de temperatura deve ser observado durante o dia, independentemente do fundo genético, uma vez que mostramos que as moscas w1118, yw e Canton S exibem uma preferência de ...

Watch this Scientific Journal Video about Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila at JoVE.com

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Recentemente identificamos uma nova produção circadiana Drosophila, ritmo de preferência de temperatura (TPR), em que a temperatura preferida em moscas sobe durante o dia e cai durante a noite. O TPR é regulado independentemente de outra produção circadiana, atividade locomotor. Aqui descrevemos o desenho e análise do TPR em Drosophila.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

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Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Unit 1

Alternative Signaling Routes in Gene Expression

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8.0K visualizações.  Cincinnati Childrens Hospital Medical Center. Recentemente identificamos uma nova produção circadiana Drosophila, ritmo de preferência de temperatura (TPR), em que a temperatura preferida em moscas sobe durante o dia e cai durante a noite. O TPR é regulado independentemente de outra produção circadiana, atividade locomotor. Aqui descrevemos o desenho e análise do TPR em Drosophila.

Vídeo: Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolução de Medição do Comportamento Odor-Driven em larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biologia

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Transplante de lente em Zebrafish e sua Aplicação na Análise de Mutantes Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissecção do giro denteado do hipocampo de rato adulto

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Estudar checkpoint mitótico por Ilustrando comportamento dinâmico cinetocoro Proteína e Movimento de cromossomos em Viver<em&gt; Drosophila</em&gt; Os embriões Sincicial

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Extração e Análise de Cortisol de cabelo humano e do macaco

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Citológica Análise da espermatogênese: Preparados ao vivo e fixo<em&gt; Drosophila</em&gt; Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Rápida análise de fenótipos circadianos em Arabidopsis Protoplastos transfectadas com um relógio Reporter luminescentes

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Observação e quantificação do comportamento de acasalamento no nemátodo do pinheiro,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Análise do comportamento de crescimento de Arabidopsis thaliana em diferentes qualidades de luz

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...