Nicotiana benthamiana plantas são cultivadas em câmara de crescimento com temperatura controlada a 25 ° C. Totalmente expandido folhas saudáveis ​​de 3-6 semanas de idade as plantas são utilizadas. Dica: Melhores resultados são obtidos usando as folhas emergentes 1. Agrobacterium protocolo de infiltração transitória: 1 dia <ol><li> LB raia / rifampicina (25 mg / ml) / canamicina (100 mg / ml) placas de agar com ações de glicerol de Agrobacterium tumefaciens (estirpe LBA4404) contendo os vetores apropriados com o gene (s) a ser testada para a morte celular eo vetor contendo a cassete GUS sob um promotor constitutivo. Sempre incluir um controle do vetor vazio como controle negativo. </li><li> Incubar a 28 ° C por 2 dias. </li></ol> Dia 3 <ol><li> Inocular 2 ml de LB contendo rifampicina (25 mg / ml) e canamicina (100 mg / ml), com uma única colônia de cada LB / rifampicina / canamicina placa previamente riscado. </li><li> Incubar cada cultura por agitação a 28 ° C durante 24 horas a 200 rpm até a densidade máxima de crescimento é atingida. </li></ol> Dica: A aglomeração é por vezes observada, caso em que as culturas precisam ser cuidadosamente ressuspendido (ou seja, pipetando para cima e para baixo) antes de prosseguir. Dia 4 <ol><li> Após a incubação, aumentar o volume total de 10 mL com LB fresco (contendo os antibióticos apropriados) e acetosyringone (final de concentração 25 mM). </li><li> Incubar cada cultura por agitação a 28 ° C por mais 16 hrs. </li></ol> Dia 5 <ol><li> No dia seguinte, lave duas vezes cada cultura pela adição de 10 ml (não adicionar acetosyringone) médio de infiltração (10 mM MgSO 4 .7 H 2 O, 9 mM MES, pH 5,6) e centrifugar a 4000 xg por 10 min em temperatura ambiente. </li><li> Após a etapa de centrifugação final, ressuspender cada cultura em 5 ml contendo meio de infiltração acetosyringone (100 mM). </li><li> Medir a 600nm OD, medidas entre 0,1 e 0,9 são aceitáveis. Dica: OD 600nm de tão baixo quanto 0,1 têm sido usados ​​sem problema, no entanto, maior ODs pode produzir resultados mais consistentes. </li><li> Incubar as culturas de mais 3 horas para preparar as culturas Agrobacterium para a infecção. </li><li> Mix culturas contendo o gene (s) para ser analisado e do controle negativo (Agrobacterium contendo o vetor vazio) na proporção de 1:1 com a cultura contendo o cassete de GUS. </li><li> Infiltrar-se na lateral (sob) abaxial de folhas emergentes recém usando uma agulha de 1 ml menos seringa (Fig. 1). Dica: Infiltrate tanto a mistura de controle negativo (vetor vazio + cassete GUS) ea mistura contendo o gene a ser testada (gene x + cassete GUS) em folhas opostas da mesma planta. Use plantas em triplicado para cada tratamento. </li><li> Três dias pós-consumo infiltração infiltrado folhas e ensaio para a expressão da proteína GUS. </li></ol> 2. Ensaio histoquímico GUS Dia 8 <ol><li> Vácuo se infiltrar no meio do substrato X-glic nas folhas excisadas. </li><li> Incubar no escuro à temperatura ambiente durante a noite ou até coloração azul aparece distintas. </li></ol> Dia 9 <ol><li> Enxágüe em água destilada. </li><li> Incubar em etanol 70% até a clorofila é removido, então a transferência para água destilada novamente. Visualmente avaliar os níveis de expressão GUS. </li></ol> 3. MUG ensaio fluorimétrico: Dia 8 <ol><li> Isolar proteína total por trituração discos de folhas infiltradas em um almofariz com nitrogênio líquido. Adicione 100 uL de tampão de extração GUS (50 mM pH 7,0 NAPI, 10 mM EDTA, 0,1% Triton X-100, 0,1% N-lauroylsarcosine sal de sódio, β-mercaptoetanol (0,7 mL / ml), mantendo a amostra em gelo. </li><li> Centrifugar a 14.000 g de suspensão por 5 min a 4 ° C e tomar sobrenadante como proteína solúvel total. </li><li> Medir a concentração usando um nanodrop de acordo com instruções do fabricante. </li><li> Ajustar a concentração de proteína e 100 mg de proteína solúvel total para cada amostra usando GUS tampão de extração. </li><li> Adicionar substrato MUG (4-metil-β-D-glicuronídeo hidratado) preparado em GUS tampão de extração para uma concentração final de 2 mM. Um volume total de 100 mL por amostra (proteína + substrato MUG) deve ser suficiente. </li><li> Incubar as reações de 1 hora a 37 ° C. </li><li> Parar reações adicionando 800 mL de GUS batente do amortecedor (0,2 M Na 2 CO 3). </li><li> Medida de fluorescência usando um leitor de placas em um comprimento de onda de excitação de 365 nm e comprimento de onda de emissão de 455 nm e comparar os valores de uma curva padrão de MU. Níveis relatório de actividades do GUS como nmol total de MU / mg proteína solúvel / min. </li></ol> 4. Ações e Soluções: <ol><li> Acetosyringone (1 M) de estoque (FW = 196,2 g) </sub&gt; - 0,981 g em 5 mL de DMSO </li><li> Canamicina (100 mg / ml) de ações - preparar em dH 2 0 - filtro de esterilizar </li><li> Rifampicina (25 mg / ml) de ações - preparar em DMSO </li><li> Luria-Bertani (LB) líquido meios de cultura: (1L) 1% (w / v) Bacto-triptona, 10 g 0,5% (w / v) extrato de levedura-Bacto, 5 g 170 mM cloreto de sódio 10 g pH - 7 </li><li> Acetosyringone (25 mM) = 25 mL de um estoque M 1 em um volume final de 1 ml </li><li> Médias de infiltração: (1L) 10 mM MgSO 4 0,7 dH 2 0 (FW = 246,48 g), 2,4648 g 9 mM MES (PF = 213,25 g), 1,91925 g pH - 5.6 </li><li> Acetosyringone (100 M) = 100 mL de um estoque 1M em um volume final de 1 ml </li><li> GUS Extração de buffer 50 mM NAPI pH 7,0, 10 mM EDTA, 0,1% Triton X-100, 0,1% N-lauroylsarcosine sal de sódio, β-mercaptoetanol (0,7 mL / ml) </li><li> 2x tampão fosfato 0.2M NaH 2 PO 4 e 0,2 M Na 2 HPO 4 pH 7 </li><li> X-Glic solução de substrato Dissolver 1 mg de 5-bromo-4-cloro-3-indolil bD-glicuronídeo (X-Glic) em 0,1 ml de metanol. Adicionar 1 ml de tampão fosfato 2x, 20 de potássio 0,1 M mL de ferricianeto, 10μl Triton X-100 10% e 850 mL de água destilada. </li></ol> 5. Resultados representativos: Na sequência deste protocolo, perda de expressão GUS é esperada quando ocorre a morte celular. Temos, simultaneamente, expressa o gene repórter, β-glucuronidase (GUS) com um membro da Arabidopsis cito-protetora Bcl-2 Associated athanoGene (BAG) da família. Nós co-infiltradas N. benthamiana folhas com um cassete de expressão impulsionado BAG 35S eo GUS vector pCAMBIA 2301 usando Agrobacterium LBA4404 tensão. Como mostrado na figura 2, um aumento visível de coloração GUS foi observada após essa infiltração. Inversamente, quando o conhecido membro pró-apoptótico da família Bcl-2 BAX foi usado, uma redução acentuada de coloração GUS foi observada (Fig. 2). Em ambos os casos, a expressão de GUS foi visivelmente diferente se comparado ao controle. No entanto, quando a diferença de expressão é menos evidente, ensaios MUG fluorimétricos pode ser realizada para quanitate expressão GUS. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Figura 1"/> Figura 1. Exemplo de Agrobacterium infiltração mistura do lado abaxial de N. benthamiana folhas usando uma seringa de 1 ml com agulha menor. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Figura 2"/> Figura 2. Um vetor GUS foi co-expresso em N. benthamiana folhas com o gene anti-morte, e um conhecido indutor de morte celular. Níveis de GUS foram comparados com um controle do vetor vazio (GUS controle).

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, 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=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, 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> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

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<p class="jove_content"&gt; Muitas vezes, é difícil de usar técnicas de detecção de células morte em plantas que são comuns em sistemas de mamíferos. Em combinação com um sistema de repórter GUS, apresentamos uma planta de método, com base sensível para a detecção e análise de jogadores morte celular. Este método aproveita o simples fato de que as células vivas são necessários para o GUS expressão a ocorrer. Para garantir resultados significativos e repetibilidade, é fundamental que as culturas abrigar a cassete GUS eo gen...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Rifampicina </td><td> VWR </td><td> IC19549001 </td><td> 25mg/ml estoque em DMSO </td></tr><tr><td> 4&#39;-hidroxi-3 &#39;, 5&#39;-dimethoxyacetophenone (Acetosyringone) </td><td> VWR </td><td> TCD2666 </td><td> 0,981 g / mL em DMSO (1M estoque) </td></tr><tr><td> Monohidrato do ácido (4-morfolino) ethanesulfonic (MES) - 2 </td><td> VWR </td><td> EM-6110 </td><td> 1,92 g / L na água para fazer um L de mídia infiltração </td></tr><tr><td> Bacto-triptona </td><td> Pescador </td><td> BP1421-2 </td><td> 10g / L na água para fazer 1 L de meio LB </td></tr><tr><td> Bacto levedura-extrato de </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5g / L na água para fazer 1 L de meio LB </td></tr><tr><td> Cloreto de sódio </td><td> VWR </td><td> EM-7710 </td><td> 10g / L na água para fazer 1 L de meio LB </td></tr><tr><td> Fosfato de sódio monobásico </td><td> VWR </td><td> MK-7868-12 </td><td> 2,5 g / L em água para fazer 50mM de tampão NAPI </td></tr><tr><td> Fosfato de sódio dibásico hepta </td><td> VWR </td><td> EMD-SX0715-1 </td><td> 5g / L na água para fazer 50mM de tampão NAPI </td></tr><tr><td> Sulfato de magnésio hepta-hidratado </td><td> VWR </td><td> EM-MX0070-1 </td><td> 2,5 g / L na água para fazer um L de mídia infiltração </td></tr><tr><td> N-Lauroylsarcosine </td><td> VWR </td><td> TCL0151-500G </td><td> 0,1% v / v em tampão GUS </td></tr><tr><td> 5-Bromo-4-cloro-3-indoxil-beta-D-glicuronídeo sal cyclohexylammonium (X-glic) </td><td> Biotecnologia ouro </td><td> G1281C </td><td> 1mg/100uL em metanol 100% </td></tr><tr><td> 4-metil-D-μ-glicuronídeo hidratado (MUG) </td><td> Sigma </td><td> M5664 </td><td> 2 mM em 100 uL de tampão de extração GUS </td></tr><tr><td> Trihidrato de ferrocianeto de potássio </td><td> VWR </td><td> EM-PX1460-1 </td><td> Ações 100mM para X-glic solução de substrato </td></tr><tr><td> β-Methylumbelliferone (MU) </td><td> Sigma-Aldrich </td><td> M1381 </td><td> Para a curva MU tampão uso padrão de extração GUS </td></tr><tr><td> Carbonato de sódio </td><td> VWR </td><td> EM-SX0395-11 </td><td> 0,2 M em água para fazer GUS tampão parar </td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

Nós desenvolvemos um sistema de expressão transiente romance planta que ao mesmo tempo expressa o gene repórter, β-glucuronidase (GUS), com supostos reguladores positivos ou negativos de morte celular. Neste sistema, N. deixa benthamiana são co-infiltradas com uma cassete de expressão 35S impulsionado contendo o gene a ser analisado, eo GUS vector pCAMBIA 2301 usando Agrobacterium LBA4404 tensão como um veículo. Porque as células vivas são necessários para a expressão GUS a ocorrer, a perda da atividade GUS é esperada quando este gene marcador é co-expressa com os reguladores positivos da morte celular. Igualmente, o aumento da atividade GUS é observada quando anti-apoptóticos genes são usados ​​em comparação com o controle de vetores. Como mostrado abaixo, temos utilizado com sucesso este sistema em nosso laboratório para analisar os dois jogadores pró e anti-morte. Estes incluem a planta anti-apoptótico Bcl-2 Associated athanoGene (BAG) da família, bem como, conhecidos indutores de morte celular de mamíferos, como BAX. Além disso, temos utilizado este sistema para analisar a função da morte de truncamentos específicos dentro de proteínas, o que poderia fornecer pistas sobre a modificação pós-translacional possível / ativação destas proteínas. Aqui, apresentamos um método rápido e sensível de plantas com base, como um passo inicial na investigação da morte de função de genes específicos.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Ensaios de morte celular programada comumente usado em sistemas de mamíferos, tais como DNA laddering ou ensaios de TUNEL, muitas vezes são difíceis de reproduzir em plantas. Em combinação com um sistema de repórter GUS, propomos uma rápida, planta ensaio baseado transitória para analisar as propriedades morte potencial de genes específicos.

A β-glucuronidase (GUS) Ensaio Baseado Morte celular

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

a-glucuronidase-gus-based-cell-death-assay

Research

JoVE Journal

Biology

A &#946;-glucuronidase (GUS) Based Cell Death Assay

28.0K visualizações.  Texas A&M University. Ensaios de morte celular programada comumente usado em sistemas de mamíferos, tais como DNA laddering ou ensaios de TUNEL, muitas vezes são difíceis de reproduzir em plantas. Em combinação com um sistema de repórter GUS, propomos uma rápida, planta ensaio baseado transitória para analisar as propriedades morte potencial de genes específicos.

Vídeo: A &#946;-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Estudando Biogenesis de membrana com um ensaio de luciferase Gene-Based Reporter

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biologia

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificado anexina V / propídio Assay Apoptose Iodeto para avaliação rigorosa da morte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificado embrionárias de camundongos com células-tronco Ensaio baseado para quantificar a eficiência de indução cardiogênico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Um ensaio simples repórter baseado em fluorescência para identificar componentes celulares necessários para toxina ricina uma cadeia (RTA) o tráfico de levedura

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

O ensaio baseado em microscopia para estudar e analisar os endossomos de reciclagem usando o tráfico de SNARE

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Um método passo-a-passo para detectar anticorpos neutralizantes contra a AAV usando um ensaio baseado em células coloridas

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Noções Básicas de Histologia e Detecção de Morte Celular em Tecido de Abelhas Melíferas

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Extensão da vida útil dos neutrófilos com CLON-G e ensaio de morte espontânea in vitro

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

Quantificando o início da morte celular por meio de imagens contínuas

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Autor em destaque: uma abordagem simplificada para estudar a iniciação da morte celular na resposta de hipersensibilidade 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Estudando a iniciação da morte celular usando um microscópio digital

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...