Nicotiana benthamiana Pflanzen werden in einem temperierten Klimakammer bei 25 ° C gewachsen. Voll ausgebaut gesunde Blätter von 3-6 Wochen alten Pflanzen verwendet werden. Tipp: Bessere Ergebnisse werden mit neu entstehenden Blättern gewonnen 1. Agrobacterium vorübergehende Infiltration Protokoll: Tag 1 <ol><li> Streak LB / Rifampicin (25 ug / ml) / Kanamycin (100 ug / ml) Agarplatten mit Glycerin Aktien von Agrobacterium tumefaciens (Stamm LBA4404) mit den entsprechenden Vektoren mit dem Gen (s) zum Zelltod untersucht werden und der Vektor mit den GUS-Kassette unter einem konstitutiven Promotor. Enthalten immer eine leere Vektor-Regelung als negative Kontrolle. </li><li> Inkubation bei 28 ° C für 2 Tage. </li></ol> Tag 3 <ol><li> Inoculate 2 ml LB mit Rifampicin (25 ug / ml) und Kanamycin (100 ug / ml) mit einer einzelnen Kolonie von jeder LB / Rifampicin / Kanamycin Platte zuvor gestreift. </li><li> Inkubieren jede Kultur durch Schütteln bei 28 ° C für 24 Stunden bei 200 Umdrehungen pro Minute, bis ein maximales Wachstum Dichte erreicht ist. </li></ol> Tipp: Horstig ist manchmal zu beobachten, in welchem ​​Fall Kulturen gründlich resuspendiert werden (dh Auf-und Abpipettieren), bevor Sie fortfahren müssen. Tag 4 <ol><li> Nach der Inkubation erhöht das Gesamtvolumen auf 10 ml mit frischem LB (mit den entsprechenden Antibiotika) und Acetosyringon (Endkonzentration 25 uM). </li><li> Inkubieren jede Kultur durch Schütteln bei 28 ° C für weitere 16 Stunden. </li></ol> Tag 5 <ol><li> Am nächsten Tag waschen jede Kultur zweimal durch Zugabe von 10 ml (fügen Sie keine Acetosyringon) Infiltration Medium (10 mM MgSO 4 .7 H 2 O, 9 mM MES, pH 5,6) und zentrifugieren bei 4000 xg für 10 min bei Raumtemperatur. </li><li> Nach dem letzten Zentrifugationsschritt resuspendieren jede Kultur in 5 ml Medium mit Infiltration Acetosyringon (100 uM). </li><li> Messen Sie die OD 600nm, werden die Messungen zwischen 0,1 und 0,9 zulässig. Tipp: OD 600nm von nur 0,1 ohne Probleme verwendet wurden, können jedoch auch höhere OD produzieren mehr konsistente Ergebnisse. </li><li> Inkubieren Sie die Kulturen weitere 3 Stunden auf die Agrobacterium Kulturen zur Infektion vorzubereiten. </li><li> Mix Kulturen, die das Gen (s) zu analysieren und die Negativ-Kontrolle (Agrobacterium mit dem leeren Vektor) in einem Verhältnis von 1:1 mit der Kultur, die das GUS-Kassette. </li><li> Infiltrate der abaxialen (unter-) Seite neu entstehenden Blätter mit einer 1 ml nadellose Spritze (Abb. 1). Tipp: Infiltrate sowohl die Negativ-Kontrolle-Gemisch (Leervektor + GUS-Kassette) und die Mischung, die das Gen zu untersuchenden (Gen x + GUS-Kassette) auf gegenüberliegenden Blätter der gleichen Pflanze. Verwenden Sie dreifach Pflanzen für jede Behandlung. </li><li> 3 Tage nach Infiltration Verbrauchsteuern infiltriert Blätter und Assay für GUS-Protein-Expression. </li></ol> 2. Histochemische GUS-Test Tag 8 <ol><li> Vacuum infiltrieren die X-Gluc Trägermedium in die ausgeschnittene Blätter. </li><li> Inkubieren im Dunkeln über Nacht bei Raumtemperatur oder bis deutliche Blau-Färbung erscheint. </li></ol> Tag 9 <ol><li> Spülen in destilliertem Wasser. </li><li> Inkubieren in 70% Ethanol bis Chlorophyll entfernt wird, dann Transfer zum destilliertem Wasser wieder. Optisch bewerten GUS-Expression Ebenen. </li></ol> 3. Fluorometric MUG-Assay: Tag 8 <ol><li> Isolieren gesamten Proteins durch Schleifen infiltriert Blattscheiben in einem Mörser mit flüssigem Stickstoff. Add 100 uL der GUS-Extraktionspuffer (50 mM NaPi pH 7,0, 10 mM EDTA, 0,1% Triton X-100, 0,1% N-Lauroylsarcosin Natriumsalz, β-Mercaptoethanol (0,7 ul / ml), während die Probe auf Eis. </li><li> Centrifuge Suspension bei 14.000 g für 5 min bei 4 ° C und nehmen Überstand als Gesamt-lösliches Protein. </li><li> Measure-Konzentration mit Hilfe eines NanoDrop nach den Anweisungen des Herstellers. </li><li> Passen Sie die Protein-Konzentration auf 100 ug Gesamt-lösliches Protein für jede Probe mit GUS-Extraktionspuffer. </li><li> Add MUG Substrat (4-Methylumbelliferyl-β-D-Glucuronid Trihydrat) in GUS-Extraktionspuffer zu einer Endkonzentration von 2 mM hergestellt. Ein Gesamtvolumen von 100 mL pro Probe (Protein + MUG Substrat) sollte ausreichend sein. </li><li> Inkubieren Sie die Reaktionen für 1 Stunde bei 37 ° C. </li><li> Stop-Reaktionen durch Zugabe von 800 ul GUS Stop Puffer (0,2 M Na 2 CO 3). </li><li> Messen Sie mit einem Fluoreszenz-Reader bei einer Anregungswellenlänge von 365 nm und Emissionswellenlänge von 455 nm und Werte vergleichen zu einem MU Standardkurve. Bericht Ebenen der GUS-Aktivität als nmol MU / ug gesamte lösliche Protein / min. </li></ol> 4. Stocks and Solutions: <ol><li> Acetosyringon (1 M) Aktie (FW = 196,2 g) </sub&gt; - 0,981 g in 5 ml DMSO </li><li> Kanamycin (100 mg / ml) auf Lager - in dH vorbereiten 2 0 - Filter sterilisieren </li><li> Rifampicin (25 mg / ml) auf Lager - in DMSO vorbereiten </li><li> Luria-Bertani (LB) flüssige Nährmedien: (1L) 1% (w / v) Bacto-Trypton, 10 g 0,5% (w / v) Bacto-Hefeextrakt, 5 g 170 mM Natriumchlorid 10 g pH - 7 </li><li> Acetosyringon (25 nM) = 25 ul einer 1 M Stammlösung in einem Endvolumen von 1 ml </li><li> Infiltration Medium: (1L) 10 mM MgSO 4 .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> Acetosyringon (100 uM) = 100 ul einer 1M Lager in einem Endvolumen von 1 ml </li><li> GUS-Extraktionspuffer 50 mM NaPi pH 7,0, 10 mM EDTA, 0,1% Triton X-100, 0,1% N-Lauroylsarcosin Natriumsalz, β-Mercaptoethanol (0,7 ul / ml) </li><li> 2x Phosphatpuffer 0,2 M NaH 2 PO 4 und 0,2 M Na 2 HPO 4 pH 7 </li><li> X-Gluc Substrat-Lösung 1 mg 5-Brom-4-Chlor-3-Indolyl bD-Glucuronid (X-Gluc) in 0,1 ml Methanol. 1 ml 2x Phosphat-Puffer, 20 ul 0,1 M Kaliumferricyanid, 10 &amp;mgr; l Triton X-100 10% und 850 ul destilliertes Wasser. </li></ol> 5. Repräsentative Ergebnisse: Nach diesem Protokoll ist der Verlust der GUS-Expression zu erwarten, wenn Zelltod eintritt. Wir haben gleichzeitig das Reporter-Gen, β-Glucuronidase (GUS) mit einem Mitglied des Zyto-Schutz Arabidopsis Bcl-2 assoziierte athanoGene (BAG) Familie zum Ausdruck gebracht. Wir co-infiltriert N. benthamiana Blätter mit einem 35S angetrieben BAG Expressionskassette und die GUS-Vektor pCAMBIA 2301 unter Verwendung von Agrobacterium LBA4404. Wie in Abbildung 2 gezeigt, wurde eine sichtbare Steigerung der GUS-Färbung nach dieser Infiltration beobachtet. Umgekehrt, wenn die bekannten pro-apoptotischen Mitglied der Bcl-2 Familie BAX verwendet wurde, war eine deutliche Reduktion der GUS-Färbung beobachtet werden (Abb. 2). In beiden Fällen wurde GUS-Expression sichtbar anders im Vergleich zur Kontrollgruppe. Allerdings, wenn der Unterschied im Ausdruck weniger offensichtlich ist, kann fluorometrische MUG-Assays durchgeführt, um GUS-Expression quanitate werden. <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Beispiel Agrobacterium Mischung Infiltration der abaxialen Seite des N. benthamiana Blätter mit einer 1 ml nadellose Spritze. <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Abbildung 2"/> Abbildung 2. A GUS-Vektor wurde in N. co-exprimiert benthamiana Blätter mit dem Anti-Todes-Gen, und ein bekannter Induktor von Zelltod. GUS-Spiegel wurden in ein leeres Vector-Regelung (GUS Kontrolle) verglichen.

<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; Es ist oft schwierig, den Zelltod Nachweisverfahren in Pflanzen, die häufig in Säugetier-Systemen verwenden. In Kombination mit einem GUS-Reporter-System präsentieren wir ein auf pflanzlicher Basis, empfindliche Methode zum Nachweis und zur Analyse von Zelltod Spieler. Diese Methode nutzt die Tatsache, dass lebende Zellen für GUS-Expression auftreten, sind erforderlich. Um sicherzustellen, aussagekräftige Ergebnisse und Wiederholbarkeit, ist es entscheidend, dass die Kulturen beherbergen die GUS-Kassette und das...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Rifampicin </td><td> VWR </td><td> IC19549001 </td><td> 25mg/ml Lager in DMSO </td></tr><tr><td> 4&#39;-Hydroxy-3 &#39;, 5&#39;-Dimethoxyacetophenon (Acetosyringon) </td><td> VWR </td><td> TCD2666 </td><td> 0,981 g / mL in DMSO (1M Lager) </td></tr><tr><td> 2 - (4-Morpholino) ethansulfonsäure Monohydrat (MES) </td><td> VWR </td><td> EM-6110 </td><td> 1,92 g / L in Wasser zur Herstellung von 1 L der Infiltration Medien </td></tr><tr><td> Bacto-Trypton </td><td> Fischer </td><td> BP1421-2 </td><td> 10g / L in Wasser zur Herstellung von 1 L LB-Medium </td></tr><tr><td> Bacto-Hefeextrakt </td><td> VWR </td><td> EM1.03753.0500 </td><td> 5g / L in Wasser zur Herstellung von 1 L LB-Medium </td></tr><tr><td> Kochsalz </td><td> VWR </td><td> EM-7710 </td><td> 10g / L in Wasser zur Herstellung von 1 L LB-Medium </td></tr><tr><td> Mononatriumphosphat Monohydrat </td><td> VWR </td><td> MK-7868 bis 12 </td><td> 2,5 g / L in Wasser zur Herstellung von 50mm Puffer NaPi </td></tr><tr><td> Natriumdihydrogenphosphat Heptahydrat </td><td> VWR </td><td> EMD-SX0715-1 </td><td> 5g / L in Wasser zur Herstellung von 50mm Puffer NaPi </td></tr><tr><td> Magnesiumsulfatheptahydrat </td><td> VWR </td><td> EM-MX0070-1 </td><td> 2,5 g / L in Wasser zur Herstellung von 1 L der Infiltration Medien </td></tr><tr><td> N-Lauroylsarcosin </td><td> VWR </td><td> TCL0151-500G </td><td> 0,1% v / v in GUS-Puffer </td></tr><tr><td> 5-Bromo-4-Chlor-3-Indoxyl-beta-D-Glucuronid Cyclohexylammoniumsalz (X-Gluc) </td><td> Gold-Biotechnologie </td><td> G1281C </td><td> 1mg/100uL in Methanol 100% </td></tr><tr><td> 4-Methyl-μ-D-Glucuronid-Hydrat (MUG) </td><td> Sigma </td><td> M5664 </td><td> 2 mM in 100 uL der GUS-Extraktionspuffer </td></tr><tr><td> Kaliumferrocyanid Trihydrat </td><td> VWR </td><td> EM-PX1460-1 </td><td> 100 mM Stammlösung für X-Gluc Substrat-Lösung </td></tr><tr><td> β-Methylumbelliferon (MU) </td><td> Sigma-Aldrich </td><td> M1381 </td><td> Für MU Standardkurve verwenden GUS-Extraktionspuffer </td></tr><tr><td> Natriumcarbonat </td><td> VWR </td><td> EM-SX0395-11 </td><td> 0,2 M in Wasser zur Herstellung von GUS Anschlagpuffer </td></tr></tbody></table>

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

Wir haben eine neue vorübergehende Anlage Ausdruck System, das gleichzeitig Ausdruck der Reportergen β-Glucuronidase (GUS), mit vermeintlichen positive oder negative Regulatoren des Zelltods entwickelt. In diesem System, N. benthamiana Blätter sind mit einer 35S angetrieben Expressionskassette enthält das Gen analysiert werden co-infiltriert, und die GUS-Vektor pCAMBIA 2301 unter Verwendung von Agrobacterium LBA4404 als Vehikel. Da lebende Zellen benötigt werden für die GUS-Expression auftritt, ist der Verlust der GUS-Aktivität zu erwarten, wenn dieses Markergen wird mit positiven Regulatoren des Zelltods co-exprimiert. Ebenso erhöhte GUS-Aktivität beobachtet wird, wenn anti-apoptotischen Gene im Vergleich zu den Vektor-Steuerung verwendet werden. Wie unten gezeigt, haben wir erfolgreich dieses System in unserem Labor verwendet, um sowohl pro-und anti-death-Spieler zu analysieren. Dazu gehören die Anlage anti-apoptotischen Bcl-2 assoziierte athanoGene (BAG) Familie, sowie bekannte Säugetier-Induktoren von Zelltod, wie BAX. Darüber hinaus haben wir dieses System verwendet, um den Tod in Abhängigkeit von bestimmten Verkürzungen innerhalb Proteinen, die Hinweise auf die mögliche posttranslationale Modifikation / Aktivierung dieser Proteine ​​könnte zu analysieren. Hier präsentieren wir eine schnelle und empfindliche Pflanzen basierende Methode, als einen ersten Schritt bei der Untersuchung des Todes Funktion bestimmter Gene.

Watch this Scientific Journal Video about A ÃŽÂ²-glucuronidase GUS Based Cell Death Assay at JoVE.com

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

Der programmierte Zelltod-Assays häufig in Säugetier-Systemen wie DNA laddering oder TUNEL-Assays, sind oft nur schwer in Pflanzen zu reproduzieren. In Kombination mit einem GUS-Reporter-Systems, schlagen wir eine schnelle, auf pflanzlicher Basis transienten Assay, um das Potenzial der Tod Eigenschaften bestimmter Gene zu analysieren.

Eine β-Glucuronidase (GUS) Basierend Cell Death Assay

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 Aufrufe.  Texas A&M University. Der programmierte Zelltod-Assays häufig in Säugetier-Systemen wie DNA laddering oder TUNEL-Assays, sind oft nur schwer in Pflanzen zu reproduzieren. In Kombination mit einem GUS-Reporter-Systems, schlagen wir eine schnelle, auf pflanzlicher Basis transienten Assay, um das Potenzial der Tod Eigenschaften bestimmter Gene zu analysieren.

Serie: A β-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.

Studieren Membrane Biogenesis mit einem 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 ...

Forschung

Biologie

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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.

Geändert Annexin V / Propidiumiodid Apoptose Assay für eine genaue Beurteilung des Zelltods

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.

Geändert Maus embryonalen Stammzellen basierte Assay zur Quantifizierung von Kardiogener Induction Efficiency

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

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin Immunopräzipitation-Assay für Tissue-spezifische Gene mit Frühzeitige muriner Embryonen

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Verwendung LysoTracker zum programmierten Zelltod in Embryonen Erkennen und Differenzieren embryonalen Stammzellen

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 der globalen RNA-Synthese auf der Einzelzellebene folgende 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).

Ein einfache Fluoreszenz basierende Reporter Assay zelluläre Komponenten identifizieren erforderlich für Ricin Toxin eine Kette (RTA) Handel mit Hefe

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.

Der mikroskopiebasierte Assay zur Untersuchung und Analyse der Recycling-Endosomen mittels 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 ...

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.

Eine Schritt-für-Schritt-Methode zum Nachweis neutralisierender Antikörper gegen AAV mit einem kolorimetrischen zellbasierten Assay

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