Ce travail a été soutenu par le NCI R01 CA120659 (RS).

1. Un test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales <ol><li> Préparation des cellules tumorales et les cellules endothéliales microvasculaires: les cellules tumorales du cerveau telles que les cellules U87, les cellules de mélanome B16F1, le cancer du sein MDA-MB-435, et les cellules du côlon HCT116 ont été cultivées dans du DMEM supplémenté avec 10% de FBS et pénicilline / streptomycine (tous d&#39;Invitrogen ). Cellules endothéliales microvasculaires humaines en ligne les cellules endothéliales (HMVECs) ont été cultivées dans EBM2 moyennes (Lonza) supplémenté avec 1 ug / ml d&#39;hydrocortisone et 1 ng / ml de facteur de croissance épidermique, 10% de FBS et pénicilline / streptomycine. Les cellules ont été lavées avec du PBS à trois reprises et détaché avec 0,05% de trypsine / EDTA (Invitrogen). Après centrifugation à 2000 rpm pendant 5 min, culots de cellules ont été lavées à nouveau avec du PBS. Ensuite, le culot cellulaire ont été comptés avec un hémocytomètre. </li><li> Préparation de Matrigel: Une aliquote de facteur de croissance réduite de Matrigel (BD Bioscience) a été réchauffé à la température ambiante. Avant de complètement dégelé, il a été transféré sur la glace et son liquide a été maintenu sur la glace pendant au moins 10 min. Ensuite, 50 ul de Matrigel a été plaqué au plaques de 96 puits à un niveau horizontal qui permet le Matrigel à répartir uniformément, et incubées pendant 30 min à 37 ° C. </li><li> La formation du tube: Cellules (1-2 x 10 4) ont été re-suspendu avec DMEM sans sérum pour les cellules tumorales ou EBM-2 pour les cellules endothéliales, et chargé sur le haut de la Matrigel. Si ce test a été utilisé pour mesurer les effets de certains agents sur le développement des tubules, ces agents (stimulateurs ou inhibiteurs) ont été ajoutés dans le milieu sans sérum. Chaque groupe conditionnelle contenue 4-6 puits. </li><li> Image et analyse de données: Après incubation à 37 ° C pendant la nuit, chaque puits a été analysée directement sous un microscope. Si les tubules étaient nécessaires pour la fixation, 100 ul de formol à 10% de solution saline solution a été ajoutée en douceur et dix minutes plus tard, il était prêt pour l&#39;analyse. Sous un microscope à contraste de phase 10x, tubules dans chaque domaine ont été imagées et une moyenne de 3 à 5 tubules champs aléatoires dans chaque puits a été compté. </li></ol> 2. Les résultats représentatifs: HMVECs ont été utilisées comme contrôle positif, étant donné que ces tubules ont été développés à partir claires corps cellulaires allongées qui se connectent au réseau sous forme de polygones. Cellules B16F1, U87, et MDA-MB-435 développé tubules vasculaires semblables à ceux formés par HMVECs, mais cellules HCT116 n&#39;a pas (figure 1). Une cellule de formation du tube dose-dépendante a été testé dans les cellules U87. Comme le montre la figure 2, 10000 cellules formées tubules abandonnées. Une fois les cellules ont été doublés, un solide réseau vasculaire est comparable à ceux observés dans HMVECs. En revanche, les cellules de moins de 5000 a échoué à former un phénotype vasculaire. <img src="/files/ftp_upload/3040/3040fig1.jpg" alt="Figure 1"/> Figure 1. Formation de tubes induite par HMVECs, U87, MDA-MB-435, et cellules B16F1, mais pas les cellules HCT116. Toutes les cellules (2 x 10 4) ont été chargés sur Matrigel et incubées pendant la nuit. Tubules ont été imagées en utilisant le contraste de phase. HMVECs ont été utilisées comme contrôle positif. Un représentant de 3-5 champs a été montré. Bar: 100 um. <img src="/files/ftp_upload/3040/3040fig2.jpg" alt="Figure 2"/> Figure 2. U87 cellulaire induite par les tubules dans un certain nombre cellulaire dépendante manière. Nombres différents de cellules U87, comme indiqué dans les coins ont été utilisés pour la formation du tube. HMVECs ont été utilisés pour un contrôle positif. Bar: 100 mm.

<ol>
	<li>Shao, R., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvascular+endothelial+cells+immortalized+with+human+telomerase+catalytic+protein:+a+model+for+the+study+of+in+vitro+angiogenesis.">Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis.</a> Biochemical &#38; Biophysical Research Communications. 321, 788-794 (2004).</li><li>Ribeiro, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemostatic+properties+of+the+SV-40+transfected+human+microvascular+endothelial+cell+line+(HMEC-1).+A+representative+in+vitro+model+for+microvascular+endothelium.">Hemostatic properties of the SV-40 transfected human microvascular endothelial cell line (HMEC-1). 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+significance+of+tumor+cell-lined+vasculature+in+ovarian+carcinoma:+implications+for+anti-vasculogenic+therapy.">The clinical significance of tumor cell-lined vasculature in ovarian carcinoma: implications for anti-vasculogenic therapy.</a> Cancer Biology &#38; Therapy. 1, 661-664 (2002).</li><li>Shirakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+in+vasculogenic+mimicry+and+angiogenesis+of+inflammatory+breast+cancer+xenograft.">Hemodynamics in vasculogenic mimicry and angiogenesis of inflammatory breast cancer xenograft.</a> Cancer Research. 62, 560-566 (2002).</li><li>Ruf, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+tissue+factor+pathway+inhibitors+1+and+2+in+melanoma+vasculogenic+mimicry.">Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry.</a> Cancer Research. 63, 5381-5389 (2003).</li><li>Seftor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+interactions+of+laminin+5+gamma2+chain,+matrix+metalloproteinase-2,+and+membrane+type-1-matrix/metalloproteinase+are+required+for+mimicry+of+embryonic+vasculogenesis+by+aggressive+melanoma.">Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.</a> Cancer Research. 61, 6322-6327 (2001).</li><li>Shirakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenic+mimicry+and+pseudo-comedo+formation+in+breast+cancer.">Vasculogenic mimicry and pseudo-comedo formation in breast cancer.</a> International Journal of Cancer. 99, 821-828 (2002).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Pour ce test pour réussir, la qualité de Matrigel devrait être testée en premier. Un petit échantillon peut être obtenu à partir de BD Bioscience pour pré-lancer le test en utilisant HMVECs. Produits de lots différents peuvent afficher des qualités dissemblables dans lesquelles certains lots ne fournissent pas une condition optimale pour la formation du tube. Deuxièmement, toutes les bulles doivent être évitées lors d&#39;une aliquote de Matrigel est chargé dans des plaques 96 puits, parce que les bulles ...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11995 </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Le facteur de croissance réduite Matrigel </td><td> BD Bioscience </td><td> 47743-720 </td></tr><tr><td> EBM2 kit </td><td> Lonza </td><td> CC-3156 </td></tr><tr><td> Microscope Nikon Eclipse TS100 </td><td> Nikon </td><td></td></tr></tbody></table>

a matrigel-based tube formation assay to assess the vasculogenic activity of tumor cells

Au cours des dernières décennies, un essai de formation de tube en utilisant le facteur de croissance réduite Matrigel a été typiquement employées pour démontrer l&#39;activité angiogénique des cellules endothéliales vasculaires in vitro, 1-5. Toutefois, les preuves de plus en plus récemment, a montré que ce test n&#39;est pas limité à tester le comportement des cellules endothéliales vasculaires. Au lieu de cela, il a également été utilisé pour tester la capacité d&#39;un certain nombre de cellules tumorales pour développer un phénotype vasculaire 6-8. Cette capacité est compatible avec leur comportement vasculogenic identifiés chez des animaux xenotransplanted, un processus connu sous le nom vasculogenic mimétisme (VM) 9. Il ya une multitude de preuves démontrant que des cellules tumorales à médiation VM joue un rôle vital dans le développement tumoral, indépendant de l&#39;angiogénèse des cellules endothéliales 6, 10-13. Par exemple, les cellules tumorales ont été trouvés à participer dans le sang perfusé, la formation de canaux vasculaires dans des échantillons de tissus provenant de patients atteints de mélanome et le glioblastome 8, 10, 11. Ici, nous avons décrit ce test réseau tubulaire comme un outil utile dans l&#39;évaluation de l&#39;activité vasculogenic des cellules tumorales. Nous avons constaté que certaines lignées cellulaires tumorales, comme le mélanome B16F1 cellules, les cellules de glioblastome U87, et le cancer du sein MDA-MB-435 cellules sont capables de former des tubules vasculaires, mais certains n&#39;ont pas, comme le cancer du côlon cellules HCT116. Par ailleurs, ce phénotype vasculaire est dépendante du nombre de cellules plaquées sur le Matrigel. Par conséquent, ce test peut servir utilitaire puissant pour dépister le potentiel vasculaire d&#39;une variété de types cellulaires incluant les cellules vasculaires, les cellules tumorales ainsi que d&#39;autres cellules.

Watch this Scientific Journal Video about A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells at JoVE.com

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Un essai de formation de tube est utilisé pour évaluer l&#39;activité vasculaire des cellules tumorales.

Un test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

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65.8K vues.  University of Massachusetts. Un essai de formation de tube est utilisé pour évaluer l'activité vasculaire des cellules tumorales.

Vidéo: A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un test pour mesurer l&#39;activité des Escherichia coli Inductible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

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Biologie

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Test clonogénique: cellules adhérentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolement des cellules immunitaires des tumeurs primitives

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Une approche semi-quantitative pour évaluer la formation de biofilms utilisant le développement des colonies ridée

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

La formation de dosage souple Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Immunofluorescence quantitative pour mesurer la variation des taux de protéines au centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Essai in vitro pour mesurer Phosphatidyléthanolamine Methyltransferase activité

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

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 ...