Post-etichettatura e la misura utilizzando BD calceina AM colorante fluorescente Le cellule sono etichettati per la quantificazione dopo che hanno invaso attraverso il Matrigel BD Matrix e passato attraverso la membrana BD FluoroBlok. Di conseguenza, solo la misura finale di invasione delle cellule possono essere ottenute. <ol><li> Crescere le cellule a confluenza ~ 80%. </li><li> Preparare e reidratare il sistema di inserimento. <ol style="list-style-type:lower-alpha;"><li> Rimuovere il pacchetto di stoccaggio da -20 ° C e permettono di venire a temperatura ambiente. </li><li> Aprire la confezione ed aggiungere 500 microlitri caldo (37 ° C), mezzi di comunicazione verso l&#39;interno dei pozzi inserto. Lasciare la piastra per reidratare per 2 ore a 37 ° C, 5% di CO 2. Nota: non è necessario reidratare il patinata BD Falcon FluoroBlok 24-pozzetti sistema Inserire che sarà utilizzato come controllo la migrazione delle cellule. </li><li> Dopo la reidratazione, rimuovere con attenzione il supporto dai pozzetti inserire senza disturbare lo strato di BD Matrigel Matrix sulla membrana. Il sistema è ora pronto all&#39;uso. </li></ol></li><li> Preparare sospensioni cellulari da trypsinizing monostrati cellulari e risospendere le cellule in DMEM senza siero a 5 x 10 4 cellule / ml. Nota: Se si utilizza un diverso tipo di cellule è necessario determinare la densità ottimale di semina. Per determinare la densità di semina ottimale per il vostro tipo di cellulare su una superficie porosa crescita, utilizzare una gamma di densità di semina (cellule / cm 2) che supporti la densità di semina utilizzati su superfici non porose (es. boccette, piatti e piatti). Per esempio, se attualmente seme a 2,5 x 10 5 cellule / cm 2, seme a concentrazioni di cellule diverse tra 5 x 10 4 e 5 x 10 5 cellule / cm 2 per determinare la densità ottimale di semina iniziale. </li><li> Aggiungere 500 ml di sospensione cellulare (2,5 x 10 4 cellule) alle camere apicale. </li><li> Aggiungere 750 ml di fattore chemiotattico (5% FBS in DMEM) per ciascuna camera basale, utilizzando le porte del campione per l&#39;accesso. </li><li> Incubare il sistema BioCoat BD tumore invasione e la non patinata BD Falcon FluoroBlok 24-pozzetti Plate per 20-22 ore a 37 ° C, 5% CO 2 nell&#39;atmosfera. </li><li> Dopo l&#39;incubazione, rimuovere con attenzione di media dalle camere apicale. Questo può essere ottenuto sfogliando il contenuto in un contenitore per rifiuti. Non toccare la superficie inferiore del sistema di inserimento. </li><li> Trasferire il sistema di inserire in un secondo 24 pozzetti contenente 500 microlitri / e di 4 mcg / mL calceina AM in HBSS. Incubare per 1 ora a 37 ° C, 5% di CO 2. La soluzione calceina AM non viene rimosso dalla camera bassa prima di leggere la fluorescenza, perché è un colorante nonfluorescent vitale che viene convertito in calceina verde fluorescente dalle esterasi del citosol. </li><li> Fluorescenza delle cellule invase viene letto a lunghezze d&#39;onda di 494/517 nm (Es / Em) su un fondo di lettura lettore di piastre a fluorescenza. L&#39;impostazione di guadagno potrebbe essere necessario essere determinati empiricamente, ma un guadagno punto centrale dovrebbe essere un punto di partenza sufficiente. Un guadagno che è troppo alto può portare alla saturazione del rivelatore con il campione altamente fluorescenti, questo può impedire l&#39;acquisizione di risultati significativi. L&#39;uso di autogain (se supportato dal lettore) non è raccomandato. Un microscopio invertito a fluorescenza può essere usato per verificare i risultati, ma è particolarmente utile per fare questo la prima volta che si esegue questo test. Nota: E &#39;della massima importanza che i sistemi di inserti vengono letti utilizzando la mappa corretta piatto. Per informazioni sulle mappe di carico piatto vedere BD Bollettino tecnico n ° 436 il www.bdbiosciences.com o contattare il supporto tecnico BD (labware@bd.com). L&#39;orientamento corretto piastra è ben A1 in alto a sinistra e il logo BD Flacone orientata a destra, come il piatto viene inserito nel lettore. </li></ol> Riduzione dei dati I dati vengono espressi come nella seguente equazione: <img src="/files/ftp_upload/1475/1475eqa.jpg" alt="Equazione" /> Fondo possono essere sottratti prima del calcolo di invasione per cento delle cellule RFU = unità relative fluorescenti.

<ol>
	<li>Sethi, G., Ahn, K. S., Pandey, M. K., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol,+a+novel+triterpene,+potentiates+TNF-induced+apoptosis+and+suppresses+invasion+of+tumor+cells+by+inhibiting+NF-&#x03BA;B-regulated+gene+products+and+TAK1-mediated+NF-&#922;B+activation.">Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-&#x03BA;B-regulated gene products and TAK1-mediated NF-&#922;B activation.</a> Blood. 109, 2727-2735 (2007).</li><li>Takada, Y., Kobayashi, Y., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evodiamine+Aboloishes+Constitutive+and+Inducible+NF-&#x03BA;B+Activation+by+Inhibiting+I&#x03BA;B&#x03B1;+Kinase+Activation,+Thereby+Suppressing+NF-&#x03BA;B-regulated+Antiapoptotic+and+Metastatic+Gene+Expression,+Up-regulating+Apoptosis,+and+Inhibiting+Invasion.">Evodiamine Aboloishes Constitutive and Inducible NF-&#x03BA;B Activation by Inhibiting I&#x03BA;B&#x03B1; Kinase Activation, Thereby Suppressing NF-&#x03BA;B-regulated Antiapoptotic and Metastatic Gene Expression, Up-regulating Apoptosis, and Inhibiting Invasion.</a> J. Biol. Chem. 280, 17203-17212 (2005).</li><li>Harikumar, K. B., Kunnumakkara, A. B., Ahn, K. S., Anand, P., Krishnan, S., Guha, S., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+cysteini+residues+in+I&#x03BA;B&#x03B1;+kinase+and+NF-&#x03BA;B+(p65)+by+xanthohumol+leads+to+suppression+of+NF-&#x03BA;B-regulated+gene+products+and+potentiation+of+apoptosis+in+leukemia+cells.">Modification of the cysteini residues in I&#x03BA;B&#x03B1; kinase and NF-&#x03BA;B (p65) by xanthohumol leads to suppression of NF-&#x03BA;B-regulated gene products and potentiation of apoptosis in leukemia cells.</a> Blood. , 113-2003 (2009).</li><li>Nair, A. S., Sishodia, S., Ahn, K. S., Kunnumakkara, A. B., Sethi, G., Aggarwal, B. B. D. e. g. u. e. l. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=an+Akt+Inhibitor,+Suppresses+I&#922;B&#945;+Kinase+Activation+Leading+to+Suppression+of+NF-&#x03BA;B-Regulated+Gene+Expression,+Potentiation+of+Apoptosis,+and+Inhibition+of+Cellular+Invasion.">an Akt Inhibitor, Suppresses I&#922;B&#945; Kinase Activation Leading to Suppression of NF-&#x03BA;B-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion.</a> J. Immunol. , 177-5612 (2006).</li><li>Partridge, J., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+High-Throughput+Screening+of+Tumor+Cell+Invasion+using+a+Cell-based+Model.">Quantitative and High-Throughput Screening of Tumor Cell Invasion using a Cell-based Model.</a> Society for Biomolecular Screening 2005 Conference. , (2005).</li><li>Mannuzza, F., Flaherty, P., Ilsley, S., Kramer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+In+Vitro+Device+For+Measuring+Cell+Invasion+and+Method+for+its+Formation.">An Improved In Vitro Device For Measuring Cell Invasion and Method for its Formation.</a> US patent. , (2004).</li><li>Flaherty, P., Goldberger, A., Dery, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Fluorescent+Cell+Labeling+on+Tumor+Invasion+and+Screening+of+Anti-Metastatic+Compounds.">Effect of Fluorescent Cell Labeling on Tumor Invasion and Screening of Anti-Metastatic Compounds.</a> Mole. Biol. Cell. 12, 46a-46a (2001).</li><li>Flaherty, P., Mannuzza, F., Ilsley, S., Maliakal, J., Wu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+Anti-Metastatic+Compounds+by+a+Fluorescence+Based+Tumor+Invasion+Assay.">Screening of Anti-Metastatic Compounds by a Fluorescence Based Tumor Invasion Assay.</a> Screentech 2001 Conference. , (2005).</li><li>Mannuzza, F., Flaherty, P., Wu, M., Ilsley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Quantitative,+High-Throughput+Assay+System+for+the+Discovery+of+Anti-Cancer+Drugs.">Development of a Quantitative, High-Throughput Assay System for the Discovery of Anti-Cancer Drugs.</a> Proceedings of the Society for Biomolecular Screening. , (1999).</li></ol>

Gli autori sono dipendenti della BD Biosciences, che produce reagenti e strumenti utilizzati in questo articolo.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BD BioCoat Tumor Invasion System</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354165 or 354166</td>
<td>&nbsp;</td>
<tr>
<td>HT-1080 and NIH/3T3 cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium (DMEM; serum-free)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline, without calcium and magnesium (DPBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon FluoroBlok 24-Multiwell Insert System to be used as a cell migration control</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351157 or 351158</td>
<td>&nbsp;</td>
<tr>
<td>5% Fetal Bovine Serum in DMEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Calcein AM Fluorescent Dye</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354216 or 354217</td>
<td>&nbsp;</td>
<tr>
<td>Hanks&#x2019; Balanced Salt Solution (HBSS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon 24-well plates for post cell invasion labeling</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351147</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescence plate reader with bottom reading capabilities</td>
<td>&nbsp;</td>
<td>PerkinElmer EnVision (R), TECAN Infinite (R), Molecular Devices SpectraMax (R), BioTek Synergy (TM), BMG LABTECH PHERAstar.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an in vitro fluoroblok tumor invasion assay

La caratteristica delle cellule metastatiche è la loro capacità di invadere attraverso la membrana basale e migrare in altre parti del corpo. Le cellule devono essere in grado di secernere sia proteasi che degradano la membrana basale e migrare in modo da essere invasiva. BD sistema Tumore BioCoat Invasion fornisce cellule con le condizioni che permettono la valutazione della loro proprietà invasive<em&gt; In vitro</em<sup&gt; 1,2</sup&gt;. Si compone di un Falcon BD FluoroBlok 24-pozzetti Inserire Piatto con una membrana di 8,0 micron, dimensioni dei pori di PET che è stato uniformemente rivestito con BD Matrigel Matrix. Questo strato uniforme di BD Matrigel Matrix funge da membrana basale ricostituita<em&gt; In vitro</em&gt; Fornire una vera e propria barriera per non invasiva cellule pur presentando una struttura adeguata di proteine ​​per lo studio invasione. Il processo di rivestimento occlude i pori della membrana, il blocco non invasiva cellule migrano attraverso la membrana. Al contrario, le cellule invasive sono in grado di staccarsi da e migrano attraverso la membrana rivestita. La quantificazione di invasione delle cellule può essere raggiunto da una pre-o post-invasione delle cellule di etichettatura con un colorante fluorescente come DiIC<sub&gt; 12</sub&gt; (3) o calceina AM, rispettivamente, e misurando la fluorescenza delle cellule invasori. Poiché la membrana BD FluoroBlok blocca efficacemente il passaggio della luce 490-700 nm a efficienza&gt; 99%, fluorescenza marcata cellule che non hanno invaso non vengono rilevati da un approccio bottom-lettura lettore di piastre in fluorescenza. Tuttavia, le cellule che hanno invaso la parte inferiore della membrana non sono più al riparo dalla sorgente di luce e vengono rilevati con il lettore di piastre rispettivi proprietari. Questo video mostra una cella di test endpoint invasione, utilizzando calceina AM per rilevare le cellule invase.

Watch this Scientific Journal Video about An In vitro FluoroBlok Tumor Invasion Assay at JoVE.com

An In vitro FluoroBlok Tumor Invasion Assay

Questo video dimostra come misurare l&#39;invasione delle cellule attraverso inserti in coltura cellulare utilizzando una metodologia basata fluorescenza.

Un In vitro FluoroBlok Tumore Assay Invasion

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

an-in-vitro-fluoroblok-tumor-invasion-assay

Research

JoVE Journal

Biology

An In vitro FluoroBlok Tumor Invasion Assay

29.0K Views.  Discovery Labware. Questo video dimostra come misurare l'invasione delle cellule attraverso inserti in coltura cellulare utilizzando una metodologia basata fluorescenza.

Video: An In vitro FluoroBlok Tumor Invasion Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Test di adesione ed invasione di Agar in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Ricerca

Biologia

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolamento di cellule primarie trofoblasto mouse e Invasion Assay trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

Applicazione di un<em&gt; In vitro</em&gt; DNA Protection Assay per visualizzare stress Mediazione Proprietà del Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Un quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Nella migrazione delle cellule in vitro e saggi Invasion

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Un test per Lateral Rigenerazione Line in Zebrafish adulti

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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.

In vitro misurare phosphatidylethanolamine metiltransferasi attività

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

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro Saggio di SUMOilazione per studiare l'attività E3 ligasi SUMO

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.