Wir erkennen Juliann Jaumotte für die Idee des Sparens auf die Volumina der Reagenzien in der ATP-Assay. Wir sind zutiefst dankbar für die hervorragende administrative Unterstützung der Maria Caruso, Deb Willson, und Jackie Farrer und an die Mylan Schule der Apotheke für die finanzielle Unterstützung für diese Studien. Dank gebührt auch den Hunkele gefürchtete Krankheiten Stiftung und die Parkinson und Bewegungsstörungen Stiftung für die finanzielle Unterstützung der primären neuronalen Studien.

Keiner der Autoren haben alle Konflikte offen zu legen.

Wir haben gefunden, dass die Signalstärke in allen drei Lebensfähigkeitstests ist linear und mit Beschichtungs-Dichte korreliert. Jedoch nicht alle Assays sind gleichermaßen empfindlich auf das 2-fache bzw. 1,5-fache Veränderungen Plattierungsdichte. Für N2a-Zellen, die Infrarot-Tests sind weniger empfindlich als die ATP-Assay, insbesondere bei niedrigeren Beschichtungsdichten. Obwohl die Infrarot-Tests sind weniger empfindlich als ATP, die DRAQ5 + Sapphire-Assays und die α-Tubulin-Assays sind in guter Übereinsti...

Die häufigste Lebensfähigkeitstest in den biologischen Wissenschaften beinhaltet Zellzahlen. Dies wird durch eine Analyse der Top (jüngste) 200 Publikationen, die in PubMed mit einem der Schlüsselwörter &quot;in vitro&quot; oder &quot;Kultur&quot; auf 2013.04.29 und 2013.04.30 erschien belegt. Von diesen Publikationen, 23,5% verwendeten Zellzahl-Assays, einschließlich der manuellen Zellzahl zählt, zählt die automatisierte Zellzahl mit Imaging-Software, und Trypanblau-Ausschluss. Die Live / Dead-Assay wurde in 1% dieser Publikationen verwendet. Die Anzahl von Publikationen mit dem MTT (3 - (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromid)-Assay für metabolische Lebensfähigkeit betrug 11%. Dieser Überblick über die Literatur zeigt auch, dass die Anzahl von Publikationen mit dem MTT-Assays, wie in Verbindung mit der Zellzahl Assays war nur 3,5%. Trotz der Trend zu einer Lebensfähigkeitstest für sich zu nutzen, die Beurteilung zelluläre Funktion in Kombination mit der Zellzahl scheint die beste Wahl für die Beurteilung der Zell integrity. Zellzahlen allein nicht ausreichend, da die verbleibenden Zellen nicht funktionell oder gesund, obwohl sie in der gut 1,2 vorhanden sind. Umgekehrt kann die funktionelle Maßnahmen wie ATP erhöhen oder verringern in Abwesenheit von parallelen Veränderungen in der Anzahl der Zellen. Die Entkopplung von Stoffwechselanzeigen von Zellzahl zeigt, dass ATP und MTT-Assays sollten niemals als alleinige Lebensfähigkeitstest verwendet werden. In dem vorliegenden Bericht werden drei Lebensfähigkeitstests, die sowohl zelluläre Strukturen und Stoffwechselfunktion Umfrage beschrieben, für eine umfassendere Sicht der zellulären Integrität als einem Test von selbst leisten können. Zwei unserer Tests erfordern eine Infrarotkamera, die Fluoreszenz misst in der 700 und 800 nm-Kanäle. Rauschen in dem Infrarot-Wellenlängen gering, was zu höheren Signal-zu-Rausch-Verhältnisse 3. Die Odyssee, die wir verwenden Imager hat einen 4,5 log Dynamikbereich und eine Bit-Tiefe von 16, Translating bis 2 16 oder 65536 Schattierungen von Infrarot. Dies kann zu einer 8-Bit-Farbbilderzeugung, die nur liefert 2 8 oder 256 Farbtöne für jede Wellenlänge gegenübergestellt werden. So hat 16-Bit-Bild feinere Auflösung. Es sei angemerkt, daß die ursprünglichen Infrarotbilder werden oft grünen (800 nm) und roten (700 nm) in veröffentlichten Berichten zur Darstellung pseudocolored werden. Odyssey Bildkameras werden häufig sowohl für die Western-Blot-und In-Cell Western 4-7 verwendet. In-Cell Western auf Formaldehyd-fixierten Zellen verwenden primären Antikörper gegen jedes Protein von Interesse und beschriften Sie sie wiederum mit Infrarot-Fluoreszenzsekundärantikörper. Diese Technik ist besonders nützlich für die Phosphorylierung Endpunkte 6 sein. In unserer In-Cell Western, färben wir feste Zellen Proteine ​​des Zytoskeletts α-Tubulin oder die neuronale Mikrotubuli-assoziierten Protein 2 (MAP2) in der 800 nm-Kanal. Diese Proteine ​​sind reich genug, um hohen Signal-zu-Rausch-Verhältnis zu erhalten. Wir unseren Platten in der 700 Fleck auchnm-Kanal für Kerne mit dem DRAQ5 Fleck und für das Cytoplasma mit der Sapphire Fleck. Sowohl die Proteine ​​des Zytoskeletts und die DRAQ5 + Sapphire Flecken spiegeln somit zelluläre Strukturen. Die dritte Lebensfähigkeitstest misst metabolische Funktion und wird als &quot;Zelltiters Glo&quot;. In diesem Luciferase-basierenden Assay werden Helligkeitswerte direkt proportional zu der ATP-Spiegel. ATP-Assays werden häufig verwendet, um lebensfähige Zellen zu quantifizieren 8-12. Einschließlich das Wort &quot;Titer&quot; im Namen des Assays ist jedoch etwas irreführend, da ATP Produktion pro Zelle als eine Funktion der Toxin-Behandlungen zu ändern und kann daher nicht immer im Verhältnis zu Zellzahl 8. ATP-Spiegel kann auch durch zirkadianen Rhythmen 13 und 14 durch Zellteilung und Zelldifferenzierung 15 betroffen sein. Dennoch ist die hier gezeigte ATP-Assay leicht durchzuführen und geeignet, da ATP ist ein robusteres Maß des metabolischenLebensfähigkeit 16-21, wenn nicht Nummer Zelle an sich. Verwendung dieses Tests als Ergänzung zu den Infrarot-In-Cell Western ergibt daher ein umfassenderes Bild der zellulären Integrität als jeder Test allein.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
	</tbody>
</table>

viability assays for cells in culture

Manuelle Zellzahlen auf einem Mikroskop sind ein empfindliches Mittel zur Bewertung zelluläre Lebensfähigkeit aber zeitaufwendig und daher teuer. Computergestützte Lebensfähigkeitstests sind teuer in Bezug auf Ausrüstung, aber schneller und objektiver als die manuelle Zellzahlen sein. Der vorliegende Bericht beschreibt die Verwendung von drei solcher Lebensfähigkeitstests. Zwei dieser Assays sind Infrarot und einem lumineszierend. Beide Infrarot-Assays basieren auf einer 16-Bit-Odyssey Imager. Eine Infrarot-Assay verwendet den Farbstoff für DRAQ5 Kernen kombiniert mit dem Saphir-Färbung für Cytosol und in den Kanal 700 nm sichtbar gemacht. Die andere Infrarot-Assay, ein In-Cell Western nutzt Antikörper gegen Proteine ​​des Zytoskeletts (α-Tubulin-oder Mikrotubuli-assoziierten Protein 2) und kennzeichnet sie in der 800 nm-Kanal. Die dritte Lebensfähigkeitstest ist eine häufig verwendete Leucht Assay für ATP, aber wir verwenden ein Viertel der empfohlenen Volumens um Kosten zu sparen. Diese Messungen sind lineare und korrelieren mit der Anzahl von cells zogen, aber unterschiedlich in der Empfindlichkeit. Alle drei Tests zu umgehen zeitraub Mikroskopie und probieren Sie die gesamte Wohl, wodurch Stichprobenfehler zu reduzieren. Schließlich können alle Assays leicht innerhalb eines Tages nach dem Ende des Experiments durchgeführt werden, so dass eine größere Anzahl von Versuchen, um in kurzen Zeiträumen durchgeführt werden. Sie beruhen jedoch alle auf der Annahme, dass die Zellzahlen im Verhältnis zur Stärke Signal nach Behandlungen, eine Annahme, die manchmal nicht erfüllt, insbesondere für zelluläre ATP bleiben. Außerdem, wenn Zellen zu erhöhen oder zu verringern in der Größe nach der Behandlung, könnte dies die Signalstärke beeinflussen, ohne die Zellzahl. Wir schließen daraus, dass alle Lebensfähigkeitstests, einschließlich Handbuch zählt, leiden unter einer Reihe von Vorbehalten, aber, dass computergestützte Lebensfähigkeitstests sind auch die anfängliche Investition wert. Mit allen drei Assays zusammen ergibt sich ein umfassendes Bild der zellulären Struktur und Funktion.

Der limitierende Faktor bei diesen Versuchen ist die Infrarot-Färbung, wie die ATP-Assay ist in relativ kurzer Dauer. Für die Infrarot-Assays, erwarten wir, dass acht 96-Well-Platten können durch die Staffelung der zwei Gruppen von je vier Platten gefärbt und gescannt werden innerhalb eines Tages (siehe Abbildung 1). Diese Schätzung geht davon aus, 20 min der Fixierung, 30 min Waschen, 30 min zu blockieren, 2 h Inkubation primärer Antikörper, gefolgt von 30 min von Waschanlagen, 1 Stunde sekundä...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

Therapeutische Verbindungen werden oft zunächst in vitro-Lebensfähigkeitstests untersucht. Blindzellzählungen durch einen menschlichen Betrachter sehr empfindlich auf kleine Änderungen in der Zellzahl, aber nicht beurteilen Funktion. Computergestützte Lebensfähigkeitstests, wie hier beschrieben, kann sowohl die Struktur und Funktion in einer objektiven Weise zu beurteilen.

Die Lebensfähigkeit Assays für Zellen in Kultur

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

Research

JoVE Journal

Biology

45.9K Aufrufe.  Duquesne University.  Therapeutische Verbindungen werden oft zunächst in vitro-Lebensfähigkeitstests untersucht. Blindzellzählungen durch einen menschlichen Betrachter sehr empfindlich auf kleine Änderungen in der Zellzahl, aber nicht beurteilen Funktion. Computergestützte Lebensfähigkeitstests, wie hier beschrieben, kann sowohl die Struktur und Funktion in einer objektiven Weise zu beurteilen. 

Serie: Viability Assays for Cells in Culture

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Zählen und Bestimmung der Lebensfähigkeit von kultivierten Zellen

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Forschung

Biologie

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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

Neurocircuit Assays für Anfällen bei Epilepsie Mutanten Drosophila</em

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays

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

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Beurteilung der mitochondrialen Funktionen und Zellviabilität in Renal Überexpression Protein Kinase C-Isoenzyme

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Gekoppelt Assays zur Überwachung Proteinrückfaltung in<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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

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

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

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

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

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Massively Parallel Reporter Assays in kultivierte Säugerzellen

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Assays für den Abbau fehlgefalteter Proteine ​​in Zellen

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...