Ce travail a été financé par une subvention du Programme de bourse de nouveau Innovateur du directeur des NIH (ED). AA a été financé en partie par des prix de premier cycle de l&#39;Institut national du cancer et la Fondation nationale des sciences.

1. Faire les vecteurs viraux Les séquences codant pour le gène d&#39;intérêt, ainsi que des contrôles positifs et négatifs, sont insérés dans pBabePuro par des méthodes traditionnelles de clonage (amplification par PCR, digestion par restriction et ligature). Il ya quatre sites de restriction sur le vecteur où l&#39;ADN peut être inséré: BamHI, SnaBI, EcoRI et Sali. <ol><li> Préparer transfection qualité plasmide ADN provenant de cellules compétentes en utilisant le kit de plasmide QIAGEN midi. </li><li> Mesurer la concentration de l&#39;ADN en utilisant un spectrophotomètre NanoDrop2000. </li></ol> 2. rétrovirus production La lignée cellulaire d&#39;encapsidation Plat-E est utilisé pour produire des retrovirus écotrope qui fournira l&#39;ADNc d&#39;intérêt à des cellules NIH 3T3. <ol><li> Établir Plat-E cultures de cellules congelées <ol><li> Dégeler rapidement les cellules Plat-E dans une ° C bain d&#39;eau 37 et transférer dans un tube de 15 ml conique. Ajouter lentement 9 ml de milieu Plat-E (Dumilieu de Eagle modifié lbecco (DMEM) + 10% de sérum bovin fœtal (FBS) + pénicilline et streptomycine). </li><li> Centrifuger le tube à 180 x g pendant 5 minutes et éliminer le surnageant. </li><li> Remettre en suspension les cellules avec 10 ml de milieu et de transfert Plat-E pour une boîte de culture de 10 cm. </li><li> Incuber les cellules dans un 37 ° C, 5% de CO 2 incubateur jusqu&#39;à ce qu&#39;elles soient confluentes à 80-90% (environ 2 jours). </li></ol></li><li> Fractionnement cellulaire <ol><li> Aspirer le milieu et laver les cellules une fois avec PBS. </li><li> Ajouter 2 ml de 0,05% de trypsine-EDTA et incuber pendant 1 min à température ambiante. </li><li> Détacher les cellules par tapotement des doigts, ajouter 10 ml de milieu Plat-E, et transférer la suspension cellulaire à un tube de 15 ml. </li><li> Centrifuger le tube à 180 x g pendant 5 minutes et éliminer le surnageant. </li><li> Reprendre les cellules avec 10 ml de milieu Plat-E et semences dans de nouveaux plats au 1: 4-1: 6 dilutions. </li></ol></li><li> Plat-E semis et transfection <ol><li> Graine 2 x 10 6cellules par boîte de culture de 10 cm en utilisant un milieu Plat-E sans antibiotiques. </li><li> Incuber les cellules O / N dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Le lendemain, transférer 300 pi d&#39;Opti-MEM dans 1,5 ml tube de centrifugeuse. </li><li> Ajouter 27 ul de polyéthylènimine (PEI), un réactif de transfection rentable 12, au tube préparé avec Opti-MEM. Mélanger doucement par tapotement des doigts et incuber pendant 5 min à température ambiante. </li><li> Ajouter 9 pg d&#39;ADN plasmidique transfection de qualité dans le tube Opti-MEM / PEI, mélanger doucement au vortex et incuber pendant 15 min à température ambiante. </li><li> Ajouter l&#39;ADN / PEI goutte à goutte complexe dans le plat Plat-E, et incuber O / N à 37 ° C, 5% de CO 2. </li><li> Le lendemain, aspirer le milieu contenant le réactif de transfection et ajouter 10 ml de milieu Plat-E frais (sans antibiotiques). </li><li> Remettre les cellules dans l&#39;incubateur. </li></ol></li></ol> 3. Les cellules NIH 3T3 et les infections <ol><li> Établir NIHCultures et Semis 3T3 <ol><li> Décongeler rapidement des cellules NIH 3T3 ° C dans un bain d&#39;eau 37 et les transférer à un tube de 15 ml. Ajouter lentement 9 ml de milieu NIH 3T3 (DMEM + 10% de FBS). </li><li> Centrifuger le tube à 180 x g pendant 5 minutes et éliminer le surnageant. </li><li> Remettre en suspension les cellules avec 10 ml de milieu et NIH 3T3 transfert à une boîte de culture de 10 cm. </li><li> Incuber les cellules dans un 37 ° C, 5% de CO 2 incubateur. Il est très important que les cellules NIH 3T3 sont toujours maintenus underconfluent (50-60%), comme la fréquence de transformation spontanée (et donc les niveaux basaux de formation de foyers) augmente une fois par culture atteint la confluence. </li><li> Seed 3 x 10 5 cellules par boîte de 10 cm et incuber O / N de l&#39;infection le lendemain. </li></ol></li><li> Infection <ol><li> Récolter le surnageant retroviral de la boîte Plat-E en utilisant une seringue jetable de 10 ml, en le filtrant à travers un filtre en nylon de taille de la membrane de 0,45 um de pores, et en le transférant dans un 15 mltube. </li><li> Ajouter 10 ml de milieu Plat-E frais aux cellules (sans antibiotiques), et les retourner à l&#39;incubateur. </li><li> Aspirer le milieu de l&#39;antenne cellulaire NIH 3T3 d&#39;être infectés, et ajouter 5 ml de milieu régulière NIH 3T3 et 5 ml de surnageant filtré contenant le virus. </li><li> Ajouter à la boîte de polybrène à une concentration de 6 ug / ml. </li><li> Incuber les cellules O / N à 37 ° C, 5% CO 2. </li><li> Le jour suivant, répétez les étapes 3.2.1 - 3.2.5 pour un second tour de l&#39;infection. </li><li> Remplacez le support contenant le virus avec un milieu régulière NIH 3T3. </li><li> Laisser les cellules NIH 3T3 à croître pendant 2-3 semaines, en remplaçant le milieu, si nécessaire. Vérifier expression de la protéine d&#39;intérêt par électrophorèse des protéines classique et immunoblot des lysats de cellules entières de plaques de réplique. </li></ol></li></ol> 4. coloration et quantification <ol><li> Coloration au cristal violet <ol><li> Aspirer le milieu NIH 3T3 et placez les platssur la glace. </li><li> Laver les plats deux fois avec PBS glacé. </li><li> Fixer les cellules avec du méthanol glacé pendant 10 min. </li><li> Retirez les plats de la glace, aspirer le méthanol, et ajouter 3 ml de solution de cristal violet à 0,5% réalisés dans 25% de méthanol (RT). </li><li> Incuber les boîtes pendant 5 min à température ambiante. </li><li> Aspirer la solution de cristal violet et rincer soigneusement le plat avec Milli-Q H 2 O jusqu&#39;au pas de couleur se détache dans le rinçage. </li><li> Les laisser sécher O / N sur une paillasse (découverts). </li></ol></li><li> Foci Quantification <ol><li> Une fois les plats sont à sec, utiliser une règle pour mesurer les collections de couleur sombre de cellules sur la plaque. Ne compter que ceux qui sont plus de 5 mm de diamètre comme «foyers». Notez le nombre de foyers et de calculer des moyennes et de l&#39;importance par rapport aux contrôles. </li></ol></li></ol>

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La FFA fournit une méthode rapide et facile pour évaluer la transformation maligne in vitro. Il se prête à la projection d&#39;un nombre relativement important de gènes candidats, et ses exigences techniques modestes rendre rentable. En outre, deux ou plusieurs gènes peuvent être co-exprimés (parfois appelé un dosage &quot;coopération&quot;) pour évaluer le potentiel tumorigène de la combinaison. Les avantages de ce test se appuient sur sa technique simple, sa facilité de quantification et de sa re...

Les cellules tumorales peuvent être distingués de leurs homologues normales par un large éventail de modifications, de profils d&#39;expression génique pour Epigenomics à des changements morphologiques et proliferatives. Parmi ces derniers, réduction de la dépendance sur le sérum, la perte de contact (densité) l&#39;inhibition, l&#39;acquisition de la prolifération indépendante de l&#39;ancrage et, finalement, la capacité de former des tumeurs lorsqu&#39;elles sont injectées dans les animaux sont des indicateurs utiles, mesurables de transformation maligne 3. Plusieurs in vitro et in vivo ont été développés pour une transformation cellulaire. Les essais in vitro pour but d&#39;identifier et de mesurer des changements dans la morphologie de culture (test de formation de mise au point), la dynamique de culture (taux de croissance, la densité de saturation) et le facteur de croissance (croissance en sérum réduite) ou un mouillage (croissance dans la gélose molle) exigences. La méthode de référence pour la détermination de la nature maligne d&#39;un type de cellule reste la formation de tumeurs (les xénogreffes) chez les animaux expérimentaux. Cependant, til a coûté et la longueur des études in vivo ne font pas toujours les justifier comme une première étape de validation ou de dépistage des oncogènes candidats. Bien qu&#39;aucun essai in vitro fournit une évaluation précise du potentiel oncogène d&#39;un gène, ils ne donnent un aperçu de potentiel oncogène qui peuvent affiner l&#39;avenir des études in vivo. L&#39;un des systèmes les plus utilisés pour l&#39;évaluation du potentiel oncogénique in vitro est la Focus Formation Assay 2. Cette approche repose sur l&#39;utilisation de fibroblastes de souris NIH 3T3, une lignée cellulaire non transformée qui montre une forte inhibition de contact. Surexpression d&#39;un oncogène résultats en perte de croissance dépendante de la densité; les cellules transformées peuvent ensuite se développer dans des couches multiples, formant &quot;foyers&quot;, facilement visualisés contre la monocouche de cellules non transformées fond. La mise au point Formation Assay, puis, mesure la capacité d&#39;un oncogène candidat à induire une transformation maligne, comme le montre la perte de contact inhibition comme un phénotype mesurable. La FFA a été utilisée pour évaluer la transformation par la surexpression de protéines kinases (par exemple Src 4, BRAF 5), des facteurs de transcription (par exemple, N-myc 6) récepteurs, G couplés à la protéine (par exemple, P2RY8 7) et GTPases (par exemple, , Ras 1), entre autres. La relative facilité de ce test, il est un bon choix qui fournira une réponse rapide et visuellement évident de savoir si la surexpression du gène est suffisante pour transformer des cellules de fibroblastes de souris NIH 3T3 in vitro. La FFA décrit dans le présent protocole utilise la lignée cellulaire d&#39;encapsidation Plat-E 8, qui fournit les protéines virales d&#39;emballage, et le vecteur rétroviral 9 pBabePuro (Addgene plasmidique 1764) pour produire des retrovirus. Après la transfection avec la construction d&#39;pBabePuro contenant le gène d&#39;intérêt, la lignée cellulaire Plat-E produira retrovirus écotrope qui peut être utilisé pour infecter des cellules NIH 3T3. Tsa méthode virale de la livraison de gènes est plus efficace que les méthodes de transfection chimique traditionnels et offre un moyen d&#39;exprimer le gène 10 durable. Une fois incorporé dans le génome de cellules NIH 3T3, la surexpression du gène d&#39;intérêt est entraîné par le virus de longues répétitions terminales (LTR), le promoteur 11. Cette expression constante peut être utilisée pour déterminer si le gène d&#39;intérêt a une activité oncogène, tel que mesuré par la formation de foyers sur les cellules NIH 3T3.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
		</tr>
	</tbody>
</table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 est une base hélice-boucle-hélice leucine fermeture à glissière (bHLHZ) facteur de transcription qui est un membre de la MYC / MAX / MAD réseau. Ce est un membre de la famille atypique de MAD 13 à 15, et il a été rapporté d&#39;être impliqués dans la carcinogenèse 16,17. Par rapport à pBabePuro (contrôle négatif) et MYC (contrôle positif), les plats NIH 3T3 où MXD3 a été surexprimé avaient significativement moins foyers (figure 1A). Les données de ...

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Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Le dosage Focus Formation fournit une méthode simple pour évaluer le potentiel de transformation d&#39;un oncogène candidat.

Concentrez Formation: Un test cellulaire pour déterminer le potentiel oncogène d&#39;un gène

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

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27.7K vues.  University of California, Davis.  Le dosage Focus Formation fournit une méthode simple pour évaluer le potentiel de transformation d'un oncogène candidat. 

Vidéo: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarqueurs dans un modèle animal pour Révéler Corrélats neuronaux, hématologiques et du comportement de stress post-traumatique

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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Médecine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Une nouvelle approche chirurgicale pour l&#39;administration intratrachéale d&#39;agents bioactifs dans un modèle de souris fœtale

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Haut-débit cytométrie en flux test cellulaire pour détecter les anticorps de la N-méthyl-D-aspartate Receptor ou récepteur de la dopamine-2 dans le sérum humain

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Une modification<em&gt; In vitro</em&gt; Invasion Test pour déterminer le rôle potentiel des hormones, des cytokines et / ou facteurs de croissance dans le cancer Médiation invasion cellulaire

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Technique Gene Transfer et<em&gt; In vitro</em&gt; Essais cellule sur la base de l&#39;étude de la perte osseuse dans les troubles musculo-squelettiques

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionnelle (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

L&#39;utilisation de l&#39;essai de formation souple Agar colonie à identifier des inhibiteurs de Tumorigénicité dans les cellules cancéreuses du sein

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE Gene Silencing: Projection d&#39;un heptamère type Petit Guide ARN Bibliothèque pour les agents thérapeutiques potentiels du cancer

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

L&#39;injection de cellules syngéniques murin de mélanome pour déterminer leur potentiel métastatique dans les Poumons

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Un plexus choroïde épithéliale Modèle base de cellules de la barrière Fluid Human Blood-céphalo-rachidien pour étudier l&#39;infection bactérienne du côté basolatéral

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...