Nous reconnaissons Juliann Jaumotte à l&#39;idée d&#39;économiser sur les volumes de réactifs dans le dosage de l&#39;ATP. Nous sommes profondément reconnaissants pour le soutien administratif superbe de Marie Caruso, Deb Willson, et Jackie Farrer et à l&#39;École de pharmacie de Mylan pour fournir un soutien financier pour ces études. Merci aussi aux maladies Hunkele redouté Fondation et le Parkinson et les troubles du mouvement de la Fondation pour le soutien financier des études primaires de neurones.

Aucun des auteurs n&#39;a de conflit de divulguer.

Nous avons trouvé que la puissance du signal dans les trois essais de viabilité est linéaire et en corrélation avec la densité de placage. Cependant, tous les tests sont également sensibles à 2 fois ou 1,5 fois les changements dans la densité de placage. Pour les cellules N2a, les analyses infrarouges sont moins sensibles que le dosage de l&#39;ATP, en particulier à des densités de plaquage inférieure. Bien que les analyses infrarouges sont moins sensibles que l&#39;ATP, les DRAQ5 + Sapphire tests et les essa...

Le test de viabilité la plus courante dans les sciences biologiques implique nombre de cellules. Ceci est démontré par une analyse des meilleurs (les plus récents) de 200 publications parues dans PubMed avec l&#39;un des mots-clés «in vitro» ou «culture» sur 29/04/2013 et 30/04/2013. Parmi ces publications, 23,5% utilisés essais de comptage de cellules, y compris les manuels nombre de cellules chiffres, le nombre de cellules automatisé compte avec un logiciel d&#39;imagerie, et exclusion au bleu Trypan. Le dosage Vivant / Mort a été utilisé dans 1% de ces publications. Le nombre de publications utilisant le MTT (3 - (4,5-diméthylthiazol-2-yl) -2,5-diphényl) test pour la viabilité métabolique était de 11%. Cette revue de la littérature montre également que le nombre de publications en utilisant des tests tels que MTT en conjonction avec les tests de comptage cellulaire n&#39;était que de 3,5%. Malgré la tendance à utiliser un test de viabilité par lui-même, l&#39;évaluation de la fonction cellulaire en combinaison avec le nombre de cellules semble le meilleur choix pour l&#39;évaluation i cellulaireNtegrity. Les comptages cellulaires par eux-mêmes ne sont pas suffisants parce que les cellules restantes ne peuvent pas être fonctionnel ou en bonne santé, même si elles sont présentes dans le puits de 1,2. A l&#39;inverse, les mesures fonctionnelles telles que l&#39;ATP peut augmenter ou diminuer en l&#39;absence de changements parallèles dans le nombre de cellules. Le découplage de lectures métaboliques du nombre de cellules suggère que l&#39;ATP et MTT essais ne doivent jamais être utilisés comme seul test de viabilité. Dans le présent rapport, trois essais de viabilité, qui sondent les structures cellulaires et la fonction métabolique sont décrits, pour une vision plus globale de l&#39;intégrité cellulaire que n&#39;importe quel test en lui-même ne peut se permettre. Deux de nos tests nécessitent un imageur infrarouge qui mesure la fluorescence dans les canaux 700 et 800 nm. Le bruit est faible dans les longueurs d&#39;onde infrarouges, entraînant une hausse des signal-bruit ratios 3. L&#39;imageur de l&#39;Odyssée que nous utilisons a une gamme dynamique de 4,5 log et une profondeur de bits de 16, Translating à 2 16 ou 65 536 nuances de l&#39;infrarouge. Cela peut être comparé à l&#39;imagerie de couleur 8 bits, qui ne donne 2 8 ou 256 nuances de couleur pour chaque longueur d&#39;onde. Ainsi, l&#39;imagerie 16 bits a une résolution plus fine. Il convient de noter que les images infrarouges d&#39;origine sont souvent pseudocolored vert (800 nm) et le rouge (700 nm) dans des rapports publiés pour la présentation. imageurs Odyssey sont couramment utilisés à la fois pour Western blot et In-Cell Westerns 4-7. In-Cell Westerns sur les cellules fixées au formaldehyde utilisent des anticorps primaires contre toute protéine d&#39;intérêt et de les étiqueter à son tour avec des anticorps secondaires fluorescents infrarouges. Cette technique est connue pour être particulièrement utile pour les points d&#39;extrémité de phosphorylation 6. Dans notre In-Cell Westerns, nous colorer les cellules fixes de protéines du cytosquelette α-tubuline ou aux microtubules neuronaux de la protéine associée à 2 (MAP2) dans le canal de 800 nm. Ces protéines sont assez abondantes pour donner rapports signal sur bruit. Nous tachons également nos assiettes dans le 700canal nm pour les noyaux avec la tache de DRAQ5 et pour le cytoplasme avec la tache de Sapphire. Tant les protéines du cytosquelette et les DRAQ5 + taches Sapphire reflètent donc des structures cellulaires. Le troisième test de viabilité de mesurer la fonction métabolique et est appelé &quot;Cell Titer Glo.&quot; Pour ce dosage à base de luciférase-, valeurs de luminescence sont en proportion directe avec les taux d&#39;ATP. Essais ATP sont couramment utilisés pour quantifier les cellules viables 8-12. Cependant, y compris le mot «titre» dans le nom de l&#39;analyse est un peu un abus de langage car la production d&#39;ATP par cellule peut changer en fonction des traitements de toxine et n&#39;est donc pas toujours en proportion du nombre de cellules 8. Les niveaux d&#39;ATP peuvent également être affectés par les rythmes circadiens 13 et par la division cellulaire 14 et la différenciation des cellules 15. Néanmoins, le dosage de l&#39;ATP montré ici est simple à réaliser et utile car l&#39;ATP est une mesure robuste métaboliqueviabilité 16-21, sinon la cellule numéro en soi. L&#39;utilisation de ce test pour compléter l&#39;infrarouge In-Cell Westerns cède donc une image plus complète de l&#39;intégrité cellulaire que tout un test seul.

<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

Le nombre de cellules manuelles sur un microscope sont des moyens sensibles à l&#39;évaluation de la viabilité cellulaire, mais prennent du temps et par conséquent coûteux. Essais de viabilité informatiques sont coûteux en termes d&#39;équipement, mais peut être plus rapide et plus objective que les comptages manuels cellulaires. Le présent rapport décrit l&#39;utilisation de ces trois essais de viabilité. Deux de ces dosages sont l&#39;infrarouge et l&#39;autre est luminescent. Les deux tests infrarouges reposent sur un imageur 16 bits Odyssey. Une analyse infrarouge utilise la tache de DRAQ5 pour les noyaux combinés avec la tache de Sapphire pour cytosol et est visualisé dans le canal 700 nm. L&#39;autre test infrarouge, un In-Cell occidentale, utilise des anticorps contre les protéines du cytosquelette (α-tubuline ou microtubules protéines associées 2) et les étiquettes dans le canal 800 nm. Le troisième test de viabilité est un dosage luminescent couramment utilisé pour l&#39;ATP, mais nous utilisons un quart de la quantité recommandée pour économiser sur les coûts. Ces mesures sont toutes linéaires et sont en corrélation avec le nombre de CElls plaqué, mais varient en sensibilité. Tous les trois essais contourner microscopie temps et déguster l&#39;ensemble bien, ce qui réduit l&#39;erreur d&#39;échantillonnage. Enfin, tous les tests peuvent facilement être achevée dans un délai d&#39;une journée de la fin de l&#39;expérience, ce qui permet un plus grand nombre d&#39;expériences à réaliser dans des délais courts. Cependant, elles reposent toutes sur l&#39;hypothèse que le nombre de cellules restent en proportion de la force du signal après les traitements, une hypothèse qui est parfois pas satisfaite, en particulier pour l&#39;ATP cellulaire. En outre, si les cellules augmentent ou diminuent en taille après le traitement, cela pourrait affecter la force du signal sans affecter le nombre de cellules. Nous concluons que tous les tests de viabilité, y compris les comptages manuels, souffrent d&#39;un certain nombre de réserves, mais que les essais de viabilité informatisés valent bien l&#39;investissement initial. En utilisant les trois dosages donne ainsi une vue d&#39;ensemble de la structure et de la fonction cellulaire.

Le facteur limitant la vitesse dans ces expériences est la coloration de l&#39;infrarouge, comme le dosage de l&#39;ATP est relativement courte en durée. Pour les tests infrarouges, nous prévoyons que huit plaques à 96 puits peuvent être colorés et analysés dans un jour par échelonnement deux lots de quatre assiettes maximum (voir la figure 1). Cette estimation suppose 20 min de fixation, 30 min de lavage, 30 min de blocage, 2 h primaire incubation d&#39;anticorps puis 30 min de lavages, 1 heure...

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

Viability Assays for Cells in Culture

Les composés thérapeutiques sont souvent d&#39;abord examiné in vitro par des tests de viabilité. Le nombre de cellules aveugles par un observateur humain peuvent être très sensibles à de faibles variations du nombre de cellules mais ne pas évaluer la fonction. Essais de viabilité informatiques, tel que décrit ici, peuvent évaluer à la fois la structure et la fonction d&#39;une manière objective.

Les analyses de viabilité de cellules en culture

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 vues.  Duquesne University.  Les composés thérapeutiques sont souvent d'abord examiné in vitro par des tests de viabilité. Le nombre de cellules aveugles par un observateur humain peuvent être très sensibles à de faibles variations du nombre de cellules mais ne pas évaluer la fonction. Essais de viabilité informatiques, tel que décrit ici, peuvent évaluer à la fois la structure et la fonction d'une manière objective. 

Vidéo: 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.

Comptage et détermination de la viabilité des cellules cultivées

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

Recherche

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.

Les dosages des Saisies en Neurocircuit Mutants épilepsie du Drosophile</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 de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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.

Évaluation des fonctions mitochondriales et la viabilité cellulaire dans les cellules rénales surexprimant la protéine 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 ...

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.

Les analyses couplées pour la surveillance protéines repliement<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.

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec 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.

Essais Reporter massivement parallèles dans des cellules de mammifères en culture

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.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

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.

Des dosages pour la dégradation des protéines mal repliées dans des cellules

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