Os autores gostariam de agradecer ao Dr. Chris Taylor (Ohio State University) para a introdução de tecnologia de raiz peluda para o nosso laboratório e as Dras. Senthil Subramanian (South Dakota State University), Juan Zhang (Ludong University, Shandong China) para melhorar o protocolo. Agradecemos também o Dr. Cheng Hao (Nanjing University Agricultura) para ajuda a fazer este vídeo. Este trabalho é apoiado em parte por concessões do DOE (DE-SC0001295), NSF (MCB-0923779) e USDA (2010-65116-20514) para OY

O protocolo a seguir foi usado para gerar plantas raiz peludo composto em um modelo de espécies de leguminosas M. truncatula. Protocolos semelhantes foram adaptados para pelo menos oito espécies de plantas 1-4. Usamos M. plantas raiz truncatula peludo composto para estudar as funções dos genes no desenvolvimento de raiz e de nódulos. O protocolo foi dividido em quatro seções: 1) materiais vegetais a preparar, 2) geração de plantas de raiz peludo composto; 3) infecção rizóbios simbiótica e 4) transgênico identificação raiz. Utilizou-se o vetor binário contendo o gene da proteína verde fluorescente (GFP) como repórter para a triagem da raiz nas plantas transgênicas composto 3. Em comparação com antibióticos baseado seleção, GFP-despistagem é rápido, fácil e barato. Em nossa construção, a ER-expressão otimizado gene GFP é conduzido pelo promotor de super ubiquitina, que tem fortes sinais GFP constitutiva em raízes transgênicas, permitindo fácil distinção entre raízes transgênicas e não transgênicas. 1. Preparação de materiais vegetais <ol><li> Sementes de M. truncautla são colhidos a partir de plantas cultivadas em uma estufa (50% de umidade relativa, 16 / 8 h claro / escuro, 22/18 ° C de temperatura dia / noite). Sementes maduras podem ser armazenados a 4 ° C. </li><li> Cerca de 100 sementes são escarificadas em 10 ml de ácido sulfúrico concentrado (95-98%, Sigma-Aldrich, MO) por 10 min com agitação periódica. </li><li> As sementes são lavadas 5-7 vezes com água esterilizada com agitação suave. </li><li> As sementes são, então, embebidos em água à temperatura ambiente por 6 horas e mantidos a 4 ° C por 36-48 horas para sincronizar a germinação. </li><li> Cerca de 20-30 sementes são espalhadas em papel de filtro úmido colocado em uma placa de Petri. Eles são mantidos a 22 ° C, com 16 / 8 h ciclo claro / escuro, e 40 μmol.m -2. Intensidade da luz s -1, por 5-6 dias. </li><li> Plântulas germinadas são transferidas para o solo e colocadas em estufa por um mês. </li></ol> 2. Geração de Plantas Root Cabeludo Composite <ol><li> Constrói binários portadores de genes de interesse foram transformados em A. rhizogenes utilizando protocolos padrão. Nós projetamos um conjunto de vetores binários contendo o gene GFP como um marcador que facilitam a triagem de tecidos transgênicos. Esses vetores contêm promotores diferentes e todos têm sites de Gateway de clonagem (Invitrogen, Carlsbad, CA) para a sobre-expressão ou silenciamento de genes alvo 3,5. </li><li> A. rhizogenes é cultivada em 50ml de meio LB com a seleção antibiótico adequado a 28 ° C durante 16-24 horas. </li><li> As bactérias são coletadas e re-suspenso para uma concentração final de OD 600 = 0,3 em uma solução nutritiva de nitrogênio livre de plantas (Tabela 1 e 6). </li><li> Para apoiar a reabilitação de raiz peludo, uma matriz esterilizados de apoio (&quot;Lã de Rocha&quot;, Hummert International, Earth City, MO) é cortado em plugs de aproximadamente 3 cm 3. Nós colocamos quatro velas em uma placa de Petri. </li><li> Um buraco é picado por cima de cada vela usando uma ponteira de pipeta para facilitar a inserção de explantes. A. rhizogenes (5 mL) é adicionado para cada plugue. </li><li> A seção de atirar com 2-3 gemas axilares é retirado por um corte oblíquo. O explante é então inserido no plugue, e cresceu a 22 ° C por cerca de três semanas. Mantemos os explantes Medicago sem molhar durante os primeiros 10 dias, então, molhá-los com 5 ml de solução de nitrogênio-livre, quando necessário. Descobrimos que a remoção do meristema apical pode diminuir a formação de raízes adventícias. </li></ol> 3. Simbiótica rizóbios (Sinorhizobium meliloti) Infecção <ol><li> Depois de três semanas, algumas raízes adventícias vão surgir a partir da lã de rocha. As plantas de raiz peludo compostos são transferidos para areia ou vermiculite (esterilizado), com ou sem a lã de rocha. Elas são cultivadas em estufa a 22 ° C, 16 / 8 h ciclo claro / escuro, e 300 μmol.m -2. S -1 de intensidade de luz para mais uma semana. </li><li> Antes de rizóbio infecção, S. meliloti 1201 é cultivado em 50 extrato de levedura-manitol ml de meio por 7 dias a 30 ° C (Tabela 2 e 6,7). </li><li> Células re-suspensão de rizóbio são diluídos para uma concentração final de OD 600 = 0,08 usando nitrogênio solução de nutrição gratuita. A suspensão de 10 ml fresca de S. meliloti foi aplicado para cada planta composto para induzir a formação de nódulos. </li></ol> 4. Identificação Root transgênicos <ol><li> Duas semanas após inoculação com rizóbio, as plantas são compostas de enraizadas pela lavagem em água. As raízes podem ser facilmente separadas das areias ou perlite na água. </li><li> Nós colocamos as raízes cabeludas sob um microscópio UV (Nikon SMZ1500, Excitação 460-500nm, 505nm Dichroic, Barreira mais de 510 Nm.). As raízes transgênicas GFP são positivas e as raízes adventícias são GFP negativo. Em seguida, recolher as raízes according o sinal de GFP, e contar os números de nódulos, medir o comprimento da raiz, e calcular a densidade da raiz lateral. As raízes GFP negativos são usados ​​como controle. </li></ol> 5. Resultado representante Em nosso experimento, as raízes do cabelo novo reciclados a partir de explantes em 2-3 semanas após o A. rhizogenes inoculação. Sob o microscópio UV, raízes transgênicas com o gene GFP mostram forte fluorescência verde (Fig. 1). A quantidade de raízes regeneradas ea parte das raízes GFP positivos dependem das condições de explantes e do ambiente de crescimento da média plants.On composto, 25% das raízes foram produzidas raízes transgênicas 3. Para aumentar a eficiência de transformação, 1) a condenação tampa de plástico, como mostrado no vídeo, é suficiente para manter a umidade na bandeja de crescimento para os primeiros dias, e só as plantas necessitam de pouca água no primeiros dias. Rega excessiva é prejudicial para a formação de raízes cabeludas, 2) a concentração de inoculante Agrobacterium é importante para a transformação. Quantidade excessiva de células não são úteis para a formação de raízes cabeludas ou formação de nódulos. Para formação de nódulos, a solução de rega precisa ser isentos de azoto, caso contrário, nódulos poucos irá se formar. As plantas de raiz peludo composto pode ser gerado usando cotilédones ou mudas entact como o material de partida. Apenas pequenas modificações do protocolo acima são ncessary para gerar raízes cabeludas de outros tecidos 8. Importante, cada raiz peludo é um evento de transformação independente. Portanto, os fenótipos observados em uma planta composta são a soma de eventos transforamtion várias que precisava ser confirmado por repetições em várias plantas compostas <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Figura 1" /> Figura 1. A. raízes transgênicas pode ser resolvido a partir de raízes cabeludas. Nódulos B. foram formadas nas plantas raiz peludo composto. Colocamos quatro semanas de idade M. plantas raiz truncatula peludo sob o microscópio UV e as raízes GFP positivos podem ser facilmente identificados. (Seta vermelha: root GFP; seta amarela: não-GFP root) <table border="1"><tbody><tr><td> Estoque </td><td> g/200ml </td><td> ml de ações / litro </td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> 12,3 </td><td> 2 </td></tr><tr><td> CaCl 2 .2 H 2 O </td><td> 14,7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 .3 H 2 O </td><td> 6,8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td> Fe Cl 3 .6 H 2 O </td><td> 0,49 </td><td> 2,5 </td></tr><tr><td> Micronutrientes </td><td> Veja abaixo </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Micronutrientes </td><td> g por litro </td></tr><tr><td> H 3 BO 3 </td><td> 0,142 </td></tr><tr><td> MnSO 4. H 2 O </td><td> 0,077 </td></tr><tr><td> ZnSO 4 .7 H 2 O </td><td> 0,1725 </td></tr><tr><td> CuSO 4 .5 H 2 O </td><td> 0,037 </td></tr><tr><td> NaMoO 4. H 2 O </td><td> 0,024 </td></tr><tr><td> CoCl 2. H 2 O </td><td> 0,0025 </td></tr><tr><td> Niso 4 </td><td> 0,001 </td></tr></tbody></table> Tabela 1. Solução de nutrição Nitogen livre <table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0.5g </td></tr><tr><td> NaCl </td><td> 0.1g </td></tr><tr><td> MgSO 4 · 7H 2 O </td><td> 0.2g </td></tr><tr><td> Extrato de levedura </td><td> 0,4 g </td></tr><tr><td> Manitol </td><td> 10g </td></tr><tr><td> pH = 6,8 </td><td></td></tr></tbody></table> Tabela 2. Extrato de levedura manitol médio (por litro)

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

Não há conflitos de interesse declarados.

Gerando planta raiz peludo composto é um método rápido e fácil para obter grandes quantidades de material transgênico para muitas outras espécies floridas. Embora este método não pode produzir sementes transgênicas, pode produzir material transgênico em algumas semanas. O método é especialmente adequado para as plantas que têm dificuldade de estabelecer culturas de tecidos ou a geração de transformantes estáveis. Ao longo dos anos, temos usado esta tecnologia para estudar as funções dos genes, as funções de promotor, microRNAs, raiz e desenvolvimento radicular lateral, defesa e resposta ao estresse abiótico, nodulação e outros processos simbióticos, respostas hormonais, perfil metabólico, perfil genético, análise proteômica e outros processos biológicos. O protocolo é robusto e replicáveis.

generation of composite plants in medicago truncatula used for nodulation assays

Semelhante ao tumerfaciens Agrobacterium, Agrobacterium rhizogenes pode transferir DNAs estrangeiros em células de plantas com base na raiz autônoma de indução (Ri) plasmídeo. A. rhizogenes podem causar a formação de raízes cabeludas sobre os tecidos da planta e forma de plantas compostas após a transformação. Sobre essas plantas compostas, algumas das raízes regeneradas são transgênicos, carregando o tipo selvagem T-DNA eo vetor binário engenharia, enquanto os tiros ainda são não-transgênico, que serve para fornecer energia e sustentar o crescimento. Estas plantas raiz peludo composto não vai produzir sementes transgênicas, mas há uma série de características importantes que fazem estas plantas compostos muito úteis na investigação de plantas. Primeiro, com uma ampla gama de hospedeiros, A. rhizogenes pode transformar muitas espécies de plantas, principalmente dicotiledôneas, permitindo que a engenharia genética em uma variedade de espécies. Segunda, A. rhizogenes infectar tecidos e explantes diretamente, sem culturas de tecidos antes da transformação é necessária para obtenção de plantas compostas, tornando-as ideais para transformar espécies de plantas recalcitrantes. Além disso, os tecidos da raiz transgênicos podem ser gerados em uma questão de semanas. Para Medicago truncatula, podemos obter as raízes transgênicas em tão curto quanto três semanas, mais rápido do que o normal transformação mergulho floral de Arabidopsis. No geral, o cabeludo raiz tecnologia das centrais composto é uma ferramenta versátil e útil para estudar as funções dos genes e raiz-relacionados fenótipos. Aqui demonstramos como as plantas de raiz peludo composto pode ser usado para estudar as interações planta rizóbio e nodulação do M. difícil de transformar as espécies truncatula.

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Demonstramos como as plantas de raiz peludo composto pode ser usado para estudar as interações planta rizóbio e nodulação na difíceis de transformar as espécies<em&gt; Medicago truncatula</em&gt;.

Geração de plantas em Composite Medicago truncatula Utilizado para Ensaios Nodulação

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

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Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

16.0K visualizações.  Donald Danforth Plant Science Center. Demonstramos como as plantas de raiz peludo composto pode ser usado para estudar as interações planta rizóbio e nodulação na difíceis de transformar as espécies Medicago truncatula.

Vídeo: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Ensaios Neurocircuit para Apreensões em mutantes de Epilepsia Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Biologia

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hibridização In Situ para a localização precisa de transcrições em Plantas

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Ensaios de viabilidade para células em cultura

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI por espectrometria de massa de imagens de Investigação de metabólitos em<em&gt; Medicago truncatula</em&gt; Os nódulos radiculares

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medindo espacial e temporal Ca<sup&gt; 2 +</sup&gt; Sinais em Arabidopsis Plantas

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

Calibração e uso de desempenho da MRM Microcoil demonstrados em raízes de truncatula medicago a 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Microdissection laser RNA-Sequenciamento para Análise de Expressão Genética Espacial e Temporal-Específica do Tecido em Plantas

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Técnicas de Microscopia para Interpretação da Colonização Fúngica em Tecidos de Plantas Micoheterotróficas e Germinação Simbiótica de Sementes

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...