Nicotiana benthamiana sont cultivés dans une chambre de croissance à température contrôlée à 25 ° C. Entièrement développé feuilles saines de 3-6 semaines vieilles plantes sont utilisées. Astuce: De meilleurs résultats sont obtenus en utilisant des feuilles naissantes 1. Protocole de l&#39;infiltration d&#39;Agrobacterium transitoires: Jour 1 <ol><li> Plaques d&#39;agar LB Streak / rifampicine (25 pg / ml) / kanamycine (100 ug / ml) avec des stocks de glycérol d&#39;Agrobacterium tumefaciens (souche LBA 4404) contenant les vecteurs appropriés avec les gène (s) à doser pour la mort de la cellule et le vecteur contenant la cassette GUS sous un promoteur constitutif. Toujours inclure un contrôle vecteur vide comme contrôle négatif. </li><li> Incuber à 28 ° C pendant 2 jours. </li></ol> Jour 3 <ol><li> Inoculer 2 ml de LB contenant de la rifampicine (25 ug / ml) et à la kanamycine (100 ug / ml) avec une seule colonie de chaque LB / rifampicine / kanamycine plaque préalablement striée. </li><li> Incuber chaque culture en secouant à 28 ° C pendant 24 heures à 200 rpm jusqu&#39;à la densité de croissance maximal est atteint. </li></ol> Astuce: agglomérante est parfois observé, dans lequel les cultures de cas doivent être soigneusement remis en suspension (par exemple aspiration et refoulement) avant de procéder. Jour 4 <ol><li> Après incubation, augmenter le volume total à 10 mL avec LB frais (contenant les antibiotiques appropriés) et acétosyringone (concentration finale de 25 pM). </li><li> Incuber chaque culture en secouant à 28 ° C pendant 16 heures. </li></ol> Jour 5 <ol><li> Le jour suivant, lavez chaque culture à deux reprises en ajoutant 10 ml (ne pas ajouter acétosyringone) moyen d&#39;infiltration (10 mM MgSO 4 .7 H 2 O, 9 mM MES, pH 5,6) et centrifuger à 4000 xg pendant 10 min à température ambiante. </li><li> Après l&#39;étape de centrifugation finale, resuspendre chaque culture dans 5 ml contenant une infiltration moyenne acétosyringone (100 uM). </li><li> Mesurer la 600nm OD, mesure entre 0,1 et 0,9 sont acceptables. Astuce: OD 600nm d&#39;aussi faible que 0,1 ont été utilisées sans problème, cependant, plus OD peut produire des résultats plus cohérents. </li><li> Incuber les cultures pour plus de 3 heures pour préparer les cultures de l&#39;infection par Agrobacterium. </li><li> Mélanger les cultures contenant le gène (s) pour être analysés et le contrôle négatif (tumefaciens contenant le vecteur vide) dans un rapport 1:1 avec la culture contenant la cassette GUS. </li><li> Infiltrer la abaxiale (sous) côté du nouveau feuilles émergentes en utilisant un 1 ml sans aiguille de seringue (Fig. 1). Astuce: infiltrer la fois le mélange contrôle négatif (vecteur vide + cassette GUS) et le mélange contenant le gène à doser (gène GUS x + cassette) sur les feuilles opposées de la même plante. Utiliser des plantes en triple pour chaque traitement. </li><li> 3 jours après l&#39;infiltration d&#39;accise infiltré feuilles et de dosage pour l&#39;expression des protéines GUS. </li></ol> 2. Histochimiques GUS test Jour 8 <ol><li> Vide infiltrer le milieu substrat X-gluc dans les feuilles excisées. </li><li> Incuber dans l&#39;obscurité à température ambiante pendant une nuit ou jusqu&#39;à coloration bleue apparaît distincte. </li></ol> Jour 9 <ol><li> Rincer à l&#39;eau distillée. </li><li> Incuber dans l&#39;éthanol à 70% jusqu&#39;à la chlorophylle est enlevé, puis transfert à l&#39;eau distillée à nouveau. Visuellement évaluer les niveaux d&#39;expression de GUS. </li></ol> 3. MUG test fluorométrique: Jour 8 <ol><li> Isoler des protéines totales par broyage disques foliaires infiltré dans un mortier à l&#39;aide d&#39;azote liquide. Ajouter 100 ul de tampon d&#39;extraction GUS (50 mM pH 7,0 NaPi, EDTA 10 mM, 0,1% de Triton X-100, 0,1% de sel de sodium de N-lauroylsarcosine, β-mercaptoéthanol (0,7 ug / ml) tout en gardant l&#39;échantillon sur la glace. </li><li> Centrifuger la suspension à 14000 g pendant 5 min à 4 ° C et de prendre surnageant protéine soluble totale. </li><li> Mesurer la concentration en utilisant un Nanodrop conformément aux instructions du fabricant. </li><li> Ajuster la concentration de protéines pour 100 ug de protéines solubles totales pour chaque échantillon en utilisant un tampon d&#39;extraction GUS. </li><li> Ajouter un substrat MUG (4-méthyl-β-D-glucuronide trihydraté) préparé dans un tampon d&#39;extraction GUS à une concentration finale de 2 mM. Un volume total de 100 ul par échantillon (protéines + substrat MUG) devrait être suffisant. </li><li> Incuber les réactions pendant 1 heure à 37 ° C. </li><li> Arrêtez réactions en ajoutant 800 ul de GUS butoir (0,2 M Na 2 CO 3). </li><li> Mesure de fluorescence en utilisant un lecteur de plaques à une longueur d&#39;onde d&#39;excitation de 365 nm et longueur d&#39;onde d&#39;émission de 455 nm et comparer les valeurs d&#39;une courbe de MU standard. Rapport du taux d&#39;activité GUS en nmol MU / ug de protéines solubles totales / min. </li></ol> 4. Stocks et solutions: <ol><li> Acétosyringone (1 M) stock (FW = 196,2 g) </sub&gt; - 0,981 g dans 5 ml de DMSO </li><li> Kanamycine (100 mg / ml) de bouillon - préparer en DH 2 0 - stériliser par filtration </li><li> La rifampicine (25 mg / ml) de bouillon - préparer dans le DMSO </li><li> Luria-Bertani (LB) milieux de croissance liquide: (1L) 1% (p / v) bacto-tryptone, 10 g 0,5% (p / v) bacto-extrait de levure, 5 g 170 mM de chlorure de sodium 10 g pH - 7 </li><li> Acétosyringone (25 uM) = 25 pi d&#39;un stock de 1 M dans un volume final de 1 ml </li><li> Moyen d&#39;infiltration: (1L) 10 mM MgSO 4 0,7 dH 2 0 (FW = 246,48 g), 2,4648 g 9 mM MES (FW = 213,25 g), 1,91925 g pH - 5,6 </li><li> Acétosyringone (100 pM) = 100 pi d&#39;un stock 1M dans un volume final de 1 ml </li><li> GUS tampon d&#39;extraction NaPi 50 mM pH 7,0, EDTA 10 mM, 0,1% de Triton X-100, 0,1% de sel de sodium de N-lauroylsarcosine, β-mercaptoéthanol (0,7 ug / ml) </li><li> Tampon phosphate 2x 0,2 M NaH 2 PO 4 et 0,2 M Na 2 HPO 4 pH 7 </li><li> Solution de substrat X-Gluc Dissoudre 1 mg de 5-bromo-4-chloro-3-indolyl bD-glucuronide (X-Gluc) dans 0,1 ml de méthanol. Ajouter 1 ml de tampon phosphate de 2x, 20 de potassium 0,1 M ul ferricyanure, 10 ul Triton X-100 de 10% et 850 pl d&#39;eau distillée. </li></ol> 5. Les résultats représentatifs: Suite à ce protocole, la perte d&#39;expression de GUS est attendue lors de la mort cellulaire survient. Nous avons simultanément exprimé le gène rapporteur, β-glucuronidase (GUS) avec un membre de l&#39;Arabidopsis cyto-protectrice de Bcl-2 Associated athanoGene (SAC) de la famille. Nous avons co-infiltrées N. benthamiana laisse avec une cassette 35S SAC conduit expression et le vecteur de GUS pCAMBIA 2301 en utilisant la souche d&#39;Agrobacterium LBA4404. Comme le montre la figure 2, une augmentation visible de la coloration GUS a été observée à la suite de cette infiltration. Inversement, lorsque l&#39;connues pro-apoptotique de la famille Bcl-2 BAX a été utilisé, une réduction marquée de la coloration GUS a été observée (Fig. 2). Dans ces deux cas, l&#39;expression de GUS était visiblement différent par rapport au témoin. Toutefois, lorsque la différence d&#39;expression est moins évident, des dosages MUG fluorométrique peut être effectuée pour quanitate expression de GUS. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Figure 1"/> Figure 1. Exemple d&#39;infiltration mélange d&#39;Agrobacterium du côté abaxiale de N. benthamiana feuilles en utilisant un 1 ml sans aiguille de seringue. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Figure 2"/> Figure 2. Un vecteur GUS a été co-exprimées dans N. benthamiana feuilles avec le gène anti-mort, et un inducteur connu de la mort cellulaire. Niveaux de GUS ont été comparés à un contrôle vecteur vide (GUS contrôle).

<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>

<p class="jove_content"&gt; Il est souvent difficile d&#39;utiliser des techniques de détection dans la mort cellulaire des plantes qui sont courants dans les systèmes mammifères. En combinaison avec un système rapporteur GUS, nous présentons une base de plantes, méthode sensible pour la détection et l&#39;analyse des joueurs de mort cellulaire. Cette méthode tire parti du simple fait que les cellules vivantes sont nécessaires pour l&#39;expression de GUS de se produire. Afin de garantir des résultats significatifs et de répétabilit...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Rifampicine </td><td> VWR </td><td> IC19549001 </td><td> Stock de 25mg/ml dans le DMSO </td></tr><tr><td> 4&#39;-hydroxy-3 &#39;, 5&#39;-diméthoxyacétophénone (acétosyringone) </td><td> VWR </td><td> TCD2666 </td><td> 0,981 g / mL dans le DMSO (1M stock) </td></tr><tr><td> 2 - (4-morpholino) monohydrate d&#39;acide éthanesulfonique (MES) </td><td> VWR </td><td> EM-6110 </td><td> 1,92 g / L dans l&#39;eau pour faire 1 litre de médias infiltrations </td></tr><tr><td> Bacto-tryptone </td><td> Fisher </td><td> BP1421-2 </td><td> 10g / L dans l&#39;eau pour faire 1 litre de milieu LB </td></tr><tr><td> Bacto-extrait de levure </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5g / L dans l&#39;eau pour faire 1 litre de milieu LB </td></tr><tr><td> Le chlorure de sodium </td><td> VWR </td><td> EM-7710 </td><td> 10g / L dans l&#39;eau pour faire 1 litre de milieu LB </td></tr><tr><td> Le phosphate de sodium monobasique monohydraté </td><td> VWR </td><td> MK-7868-12 </td><td> 2.5g / L dans l&#39;eau pour faire 50 mM de tampon NaPi </td></tr><tr><td> Le phosphate de sodium dibasique heptahydraté </td><td> VWR </td><td> EMD-SX0715-1 </td><td> 5g / L dans l&#39;eau pour faire 50 mM de tampon NaPi </td></tr><tr><td> Sulfate de magnésium heptahydraté </td><td> VWR </td><td> EM-MX0070-1 </td><td> 2,5 g / L dans l&#39;eau pour faire 1 litre de médias infiltrations </td></tr><tr><td> N-Lauroylsarcosine </td><td> VWR </td><td> TCL0151-500G </td><td> 0,1% v / v dans du tampon GUS </td></tr><tr><td> 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide sel cyclohexylammonium (X-gluc) </td><td> Or la biotechnologie </td><td> G1281C </td><td> 1mg/100uL dans le méthanol à 100% </td></tr><tr><td> 4-méthyl-μ-D-glucuronide hydrate (MUG) </td><td> Sigma </td><td> M5664 </td><td> 2 mM dans 100 uL de tampon d&#39;extraction GUS </td></tr><tr><td> Le potassium trihydraté ferrocyanure </td><td> VWR </td><td> EM-PX1460-1 </td><td> Stock de 100 mm pour une solution de substrat X-gluc </td></tr><tr><td> β-méthylumbelliférone (MU) </td><td> Sigma-Aldrich </td><td> M1381 </td><td> Pour MU tampon, utilisez courbe standard d&#39;extraction GUS </td></tr><tr><td> Le carbonate de sodium </td><td> VWR </td><td> EM-SX0395-11 </td><td> 0,2 M dans de l&#39;eau pour faire GUS butoir </td></tr></tbody></table>

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

Nous avons développé un nouveau système d&#39;expression transitoire plante qui exprime simultanément le gène rapporteur, β-glucuronidase (GUS), avec les régulateurs putatifs positifs ou négatifs de la mort cellulaire. Dans ce système, N. feuilles benthamiana sont co-infiltrée avec une cassette d&#39;expression conduit 35S contenant le gène à analyser, et le vecteur de GUS pCAMBIA 2301 en utilisant la souche d&#39;Agrobacterium LBA4404 comme un véhicule. Parce que les cellules vivantes sont nécessaires pour l&#39;expression de GUS de se produire, la perte de l&#39;activité GUS est attendue lors de ce gène marqueur est co-exprimé avec les régulateurs positifs de la mort cellulaire. De même, augmentation de l&#39;activité GUS est observée lorsque gènes anti-apoptotiques sont utilisés par rapport à la lutte antivectorielle. Comme indiqué ci-dessous, nous avons utilisé avec succès ce système dans notre laboratoire pour analyser les deux joueurs pro et anti-mort. Il s&#39;agit notamment de l&#39;usine anti-apoptotique Bcl-2 Associated athanoGene (SAC) de la famille, ainsi que, connue des inducteurs de mort cellulaire chez les mammifères, tels que BAX. De plus, nous avons utilisé ce système pour analyser la fonction mort de troncatures spécifiques au sein des protéines, ce qui pourrait fournir des indices sur l&#39;éventuelle modification post-traductionnelle / activation de ces protéines. Ici, nous présentons une méthode rapide et sensible des plantes basée, comme une première étape dans l&#39;étude des fonctions mort de gènes spécifiques.

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

Programmé dosages mort cellulaire couramment utilisés dans les systèmes mammifères tels que l&#39;ADN ou l&#39;échelonnement des essais TUNEL, sont souvent difficiles à reproduire dans les plantes. En combinaison avec un système rapporteur GUS, nous proposons un rapide, usine de dosage basé transitoires pour analyser les propriétés de mort potentielle de gènes spécifiques.

Un β-glucuronidase (GUS) dosage basé mort cellulaire

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 vues.  Texas A&M University. Programmé dosages mort cellulaire couramment utilisés dans les systèmes mammifères tels que l'ADN ou l'échelonnement des essais TUNEL, sont souvent difficiles à reproduire dans les plantes. En combinaison avec un système rapporteur GUS, nous proposons un rapide, usine de dosage basé transitoires pour analyser les propriétés de mort potentielle de gènes spécifiques.

Vidéo: 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.

Etudier biogenèse des membranes avec un Luciferase Assay-Based gène rapporteur

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

Recherche

Biologie

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.

Mise à jour annexine V / propidium Assay apoptose iodure permettant une évaluation précise de la mort cellulaire

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.

Mise à jour embryonnaires de souris des cellules souches basée Assay pour quantifier l&#39;efficacité d&#39;induction cardiogénique

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.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

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).

Un dosage Simple journaliste Fluorescence pour identifier les composants cellulaires nécessaires à la ricine toxine une chaîne (RTA) traite des levures

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.

Le test basé sur la microscopie pour étudier et analyser les endosomes de recyclage à l’aide du trafic 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.

Une méthode étape par étape pour détecter les anticorps neutralisants contre l’AAV à l’aide d’un test colorimétrique à base de cellules

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.

Principes de base de l’histologie et détection de la mort cellulaire dans les tissus d’abeilles mellifères

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.

Prolongation de la durée de vie des neutrophiles avec CLON-G et un test de mort spontanée 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 ...

Quantification de l’initiation de la mort cellulaire par imagerie continue

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.

Pleins feux sur l’auteur : Une approche simplifiée pour étudier l’initiation de la mort cellulaire dans la réponse hypersensible 

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

Étudier l’initiation de la mort cellulaire à l’aide d’un microscope numérique

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