The authors thank Mike Myles, Sam Saunders, and C.W. Elton at the ECU Multimedia &amp; Technology Services for providing assistance in video production. We acknowledge the grant support from North Carolina Biotechnology Center, Golfers against Cancer, Brody Brothers Endowment Fund, American Heart Association, and ECU/Vidant Cancer Research and Education Fund (L.V.Y. and M.J.R.).

1. Cella Cultura ferita Chiusura Assay <ol><li> Staccare le cellule dalla piastra di coltura tissutale con 0,25% di soluzione di tripsina-EDTA. Cellule pellet in un tubo da 15 ml di centrifugazione, aspirare il surnatante e risospendere le cellule in coltura. Piatto il numero appropriato di cellule in una piastra da 6 pozzetti per il 100% di confluenza in 24 ore. Nota: Le prove possono essere necessari per determinare il tempo e il numero di cellule per raggiungere il 100% di confluenza causa del tipo di cellula e la dimensione del pozzo utilizzato (ad esempio, 1 x 10 6 cellule di melanoma B16F10 vengono seminate in un pozzetto di una 6 pozzetti 24 ore prima generazione ferita). In alternativa, possono essere utilizzate piastre da 12 pozzetti o 24 pozzetti. Nota: Se disponibile, le cellule possono essere seminate in una cultura-Insert (da LLC Ibidi) su un tessuto piastra di coltura trattata rendendo la ferita pre-colato. Una cultura-inserto può proteggere la superficie della piastra di coltura tissutale da danni dalla punta della pipetta. Se una Cultura-Insert viene utilizzato passo omettere1.2. </li><li> In un ambiente sterile (tipicamente una cappa di biosicurezza) utilizzano una punta di pipetta 200 microlitri per premere saldamente contro la sommità della piastra di coltura tissutale e rapidamente fare una ferita verticale verso il basso attraverso il monostrato cellulare. Un puntale di dimensioni diverse può essere utilizzato per ridurre le dimensioni della ferita che è desiderato. Non usare una forza eccessiva contro la piastra di coltura tissutale con la punta della pipetta in quanto potrebbe danneggiare la superficie. Se la ferita è pre-fuso, questo passaggio può essere omesso. </li><li> Con attenzione aspirare i media e detriti cellulari. Aggiungere lentamente abbastanza terreni di coltura contro la parete bene a coprire il fondo del pozzo e provocare distacchi celle aggiuntive. Dopo la generazione e l&#39;ispezione della ferita una prima immagine dovrebbe essere presa. Posizionare la piastra di coltura tissutale in un incubatore a temperatura appropriata e concentrazione di CO 2 (tipicamente 37 ° C e 5% CO 2). </li><li> In diversi punti del tempo, ad esempio ogni 3 ore, rimuovere la piastra dal incubato e posizionarlo sotto un microscopio invertito per scattare una foto snapshot e per controllare la chiusura della ferita. Se microscopia time-lapse è disponibile, scattare una foto ogni tot numero di minuti (generalmente a partire con ogni 5 minuti) per qualsiasi durata di tempo in temperatura e CO 2 camera controllata. A seconda del tipo di cellula, tempo di chiusura della ferita può variare. </li><li> Per analizzare i risultati di immagini istantanee, misurare la distanza di un lato della ferita all&#39;altro utilizzando una barra di scala. Analizzare e chiaramente presente la chiusura della ferita nel tempo utilizzando un diagramma a dispersione o grafico a barre, ad esempio, la figura 1. </li><li> Per analizzare le immagini time-lapse, di importare le immagini nel software ImageJ (liberamente disponibile a http://rsb.info.nih.gov/ij/) per fare un video. Per fare questo ImageJ aperta, fare clic su File, Importa, e Sequenza d&#39;immagini. Trovare il file con le immagini; fare clic sulla prima foto nel file. Salvare video selezionando File, Salva con nome, e AVI. Cellule che migrano singoli possono essere osservati a characterize movimento direzionale. Inoltre, le caratteristiche morfologiche quali la formazione lamellipodia e bordo di uscita svincolo possono essere studiati come bene (Figura 2 e il video supplementare). </li></ol> 2. Transwell migrazione cellulare e Invasion test <ol><li> In un ambiente sterile (tipicamente una cappa di biosicurezza) staccare le cellule dalla piastra di coltura utilizzando un buffer non enzimatica dissociazione cellulare o 0,25% di soluzione di tripsina-EDTA, cellule pellet per centrifugazione, e aspirare il supporto esistente lasciando le cellule pellet. Risospendere le cellule nel siero mezzi di coltura cellulare gratuito contenente 0,1% di BSA (albumina sierica bovina). Nota: A seconda della linea cellulare indagato 0,25% tripsina-EDTA può influenzare la migrazione cellulare e dell&#39;invasione causa della scissione di vari recettori sulla superficie cellulare 17. Nota: Il numero di cellule per ml dipende dalla dimensione delle celle. Ulteriori test possono essere necessari per trovare una densità di semina che pronisce i migliori risultati. Tipicamente 1 x 10 6 cellule / ml è un buon punto di partenza per la maggior parte dei tipi di cellule quando si utilizza il 24 e Transwell inserire. Esiste una gamma di transwell dimensioni inserto e il numero di cellule aggiunte deve essere regolata per conseguenza. </li><li> Piastra 100 ml di soluzione di cellule sulla parte superiore della membrana filtrante in un inserto transwell e incubare per 10 minuti a 37 ° C e 5% di CO 2 per permettere alle cellule di stabilirsi. Nota: La particolare dimensione dei pori della membrana transwell utilizzato dipende dalla dimensione individuale delle cellule nel campione. Ad esempio, se le cellule sono troppo piccole possono passare attraverso i pori senza la necessità di facilitare la migrazione o se sono troppo grandi non possono essere introdotte attraverso i pori. Ci sono diversi inserti transwell con dimensioni dei pori che variano da 3 micron, 5 micron, e 8 micron per saggi di migrazione cellulare. Gli inserti transwell sono disponibili in commercio da aziende come Corning. Nota: Il transwell msaggio igration può essere facilmente modificato per eseguire il saggio di invasione cellulare. A tale scopo, aggiungere matrice extracellulare (ECM) materiali sopra della membrana transwell e poi aggiungere celle sopra l&#39;ECM. Ad esempio, Matrigel è stato scongelato e liquefatto in ghiaccio, e poi 30-50 ml di Matrigel viene aggiunto a un 24-ben transwell inserire e solidificato in un incubatore a 37 ° C per 15-30 minuti per formare uno strato sottile di gel. Soluzione di cellule viene aggiunto sulla parte superiore del rivestimento Matrigel per simulare invasione attraverso la matrice extracellulare. Nota: Ci sono chiare differenze tra la migrazione delle cellule transwell ed i saggi di invasione delle cellule transwell. Il saggio migrazione cellulare transwell misura la capacità chemiotattica delle cellule verso una chemio-attrattivo. Il saggio di invasione cellulare transwell, invece, misura sia la chemiotassi delle cellule e l&#39;invasione delle cellule attraverso matrice extracellulare, un processo che si trova comunemente in metastasi del cancro o sviluppo embrionale. </li><li> Usando una pipetta, molto carefully aggiungere 600 microlitri della chemio-attractant desiderato nella parte inferiore della camera inferiore in una piastra da 24 pozzetti. Aggiungere la chemio-attrattivo senza spostare l&#39;inserto transwell ed evitare la generazione di bolle. Assicurarsi che il liquido chemio-attractant nel ben inferiore a contatto con la membrana nel pozzo superiore per formare un gradiente chemiotattico. Il tempo di incubazione dipende dal tipo di cellula e la chemio-attrattore utilizzato. Nota: Ulteriori test possono essere necessari per determinare il periodo di incubazione. Nota: Per cellule aderenti, le cellule migrate legheranno verso l&#39;altro lato della membrana 1,8. La quantificazione delle cellule migrate può essere eseguita seguendo i punti 2.4 2.8 (passi 2,4-2,8 non hanno bisogno di essere eseguita in un ambiente sterile). Per le cellule non aderenti, le cellule migrate cadranno in media nella camera inferiore. Il numero di cellule migrate può essere contato utilizzando emocitometro o citofluorimetro 5. </li><li> Rimuovere l&#39;inserto transwell dalla piastra. Utilizzare un applicatore con punta di cotone come numero di volte necessario per rimuovere delicatamente il supporto e le cellule rimanenti che non sono migrati dalla parte superiore della membrana senza danneggiarlo. </li><li> Aggiungere 600-1,000 ml di etanolo al 70% in un pozzetto di una piastra da 24 pozzetti. Posizionare l&#39;inserto transwell nel etanolo al 70% per 10 minuti per consentire la fissazione delle cellule. Rimuovere transwell inserto dalla piastra a 24 pozzetti e utilizzare un applicatore con punta di cotone per rimuovere l&#39;etanolo residuo dalla parte superiore della membrana. Lasciare la membrana transwell asciugare (in genere 10-15 minuti). </li><li> Aggiungere 600-1,000 ml di 0,2% cristal violetto in un pozzetto di una piastra da 24 pozzetti e posizionare la membrana in esso per la colorazione. Incubare a temperatura ambiente per 5-10 minuti. </li><li> Rimuovere delicatamente il violetto cristallo dalla parte superiore della membrana con un puntale o cotone applicatore capovolto. Con molta attenzione, per evitare di lavare le cellule fisse, immergere la membrana in acqua distillata tante volte quanto necessario per rimuovere l&#39;eccesso di viole cristallot. Lasciare la membrana transwell ad asciugare. </li><li> Vista sotto un microscopio invertito e contare il numero di cellule in diverse condizioni di visibilità per ottenere una somma media di cellule che sono migrate attraverso la membrana verso la chemio-attrattiva e attaccati sul lato inferiore della membrana (Figura 3). </li></ol>

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The authors declare that they have no competing financial interests.

La migrazione cellulare è un aspetto importante studiare nella ricerca sul cancro e può anche essere applicato a studi guarigione sviluppo, immunologici e ferita. La coltura cellulare ferita saggio di chiusura e la migrazione delle cellule transwell e invasione saggi di rivelare le informazioni dettagliate dei comportamenti migratori delle cellule e può essere utilizzato per studiare i meccanismi molecolari della migrazione cellulare 1,2,10,14. Il nostro studio ha utilizzato questi saggi di motilità cellu...

La motilità è una caratteristica essenziale di cellule vive. La migrazione cellulare è coinvolto nella concezione della vita, lo sviluppo embrionale, la risposta immunitaria, e molti processi patologici quali metastasi del cancro e l&#39;infiammazione 1-9. Pertanto, i metodi per lo studio delle cellule comportamento migratorio sono strumenti di ricerca molto utili per una vasta gamma di discipline nelle scienze biomediche, biologia, bioingegneria, e campi correlati. Lo studio della migrazione cellulare nella ricerca sul cancro è di particolare interesse come la principale causa di morte nei pazienti affetti da cancro è correlata alla progressione metastatica. Affinché cancro di diffondersi e diffondere in tutto il corpo, le cellule tumorali devono migrare ed invadere attraverso matrice extracellulare (ECM), intravasate in circolazione sanguigna, collegare a un sito distante, e infine estravasare per formare lontano foci 1,10-12. Vari metodi biologici possono essere utilizzati per studiare questi eventi in dettaglio. La coltura cellulare ferita alla chiusura und la migrazione transwell e invasione saggi sono ampiamente utilizzati nella comunità scientifica 1,10. Questi test possono fornire i dati necessari che possono consentire una comprensione di come un particolare tipo di cellula può migrare spontaneamente o rispondere ad una chemio-attrattiva e direzionalmente migrare verso di essa. Sono stati descritti diversi fenotipi migratori. Cellule possono migrare in forma singola cella come visto in mesenchimali o movimento ameboide simile o dal movimento multicellulare etichettato migrazione collettivo o cella in streaming 13. Il metodo di trasporto utilizzato nel cellule mobili può essere facilmente osservato utilizzando la coltura cellulare saggio chiusura della ferita. Tra i numerosi modi per studiare la migrazione delle cellule, il saggio chiusura della ferita cellulare è uno dei più semplici. Questo metodo è utile per determinare la capacità di migrazione di masse di cellule intere. Quando compiuto un ulteriore passo può essere utilizzato per osservare le caratteristiche morfologiche delle cellule dell&#39;individuo durante la migrazione 14. In seguito all&#39;analisi della chiusura della ferita molti fenotipi possono essere rivelati. Misurazione della distanza chiuso nel tempo, quando si confrontano su un controllo può rivelare cambiamenti di migrazione specifica o un fenotipo migratorio alterata che era sconosciuto in precedenza. Inoltre, la formazione lamellipodium singola cella, coda di svincolo e movimento direzionale possono dare indizi su quello che potrebbe essere compromessa o rafforzata in cellule di interesse 14. La migrazione transwell e analisi d&#39;invasione possono essere usati per analizzare la capacità delle singole cellule di rispondere direzionalmente a vari chemo-attrattivi se sono chemochine, fattori di crescita, lipidi, o nucleotidi 4,5,8,15,16. Può anche valutare la capacità migratoria differenziale a causa della sovra-espressione di un recettore 1,14. Questi saggi possono anche essere utilizzati per identificare e caratterizzare i regolatori chiave della migrazione cellulare come la famiglia delle piccole GTPasi Rho 2. Dopo questi brevi e facilmente accestest sibili, la modalità di migrazione cellulare e la capacità di una cellula di invadere in una matrice 3-D possono anche essere determinate.

<table border="0" cellpadding="0" cellspacing="0" width="601">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">11995-073</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Fetal bovine serum (FBS)</td>
			<td style="width:133px;">Gemini</td>
			<td style="width:107px;">100106</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Bovine serum albumin (BSA)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:107px;">A4503-50G</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Trypsin EDTA 0.25%</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">25200-056</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Crystal violet</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:107px;">C0775-100G</td>
			<td style="width:193px;">Dissolved in water at 0.2%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell disassociation buffer</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">13151-014</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell culture incubator</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # 3145</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">SterilGARD biosafety hood</td>
			<td style="width:133px;">The Baker Company, Inc.&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # VBM-600</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">EVOS Fl inverted microscope</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # AMF-4302-US</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:169px;">Tissue culture plate</td>
			<td style="width:133px;">Becton Dickinson</td>
			<td style="width:107px;">353046</td>
			<td style="width:193px;">Catalog number varies depending on the type of culture plate</td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:169px;">Corning Transwell insert</td>
			<td rowspan="2" style="width:133px;">Fisher</td>
			<td rowspan="2" style="width:107px;">07-200-150</td>
			<td rowspan="2" style="width:193px;">Catalog number varies depending on the pore size of the membrane</td>
		</tr>
	</tbody>
</table>

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.

La chiusura dosaggio ferita e il saggio migrazione delle cellule transwell qui presentata sono stati eseguiti utilizzando cellule di topo B16F10 melanoma come sistema modello. Nel saggio chiusura della ferita, B16F10 cellule sono state seminate in una piastra di coltura tissutale da 6 pozzetti e coltivate al 100% di confluenza in 24 ore. Una ferita di circa 700 micron di larghezza è stata generata utilizzando un puntale e la chiusura della ferita (la migrazione cellulare) è stato registrato utilizzando la microscopia ...

Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

In vitro Cell Migration and Invasion Assays

Comunemente utilizzati, sono descritti metodi altamente accessibili per l&#39;esame migrazione cellulare e l&#39;invasione in vitro. Il primo metodo è il saggio chiusura della ferita cella che misura la motilità cellulare. Il secondo metodo è il transwell migrazione e invasione test che valuta la chemiotassi e la capacità invasiva delle cellule.

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 ...

in-vitro-cell-migration-and-invasion-assays

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

In vitro Cell Migration and Invasion Assays

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

136.9K Views.  East Carolina University.  Comunemente utilizzati, sono descritti metodi altamente accessibili per l'esame migrazione cellulare e l'invasione in vitro. Il primo metodo è il saggio chiusura della ferita cella che misura la motilità cellulare. Il secondo metodo è il transwell migrazione e invasione test che valuta la chemiotassi e la capacità invasiva delle cellule. 

Video: In vitro Cell Migration and Invasion Assays

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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

Saggi Neurocircuit per Sequestri in mutanti di Epilessia Drosophila</em

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

An In vitro FluoroBlok Tumor Invasion Assay

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 both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

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 ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

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

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro aggregazione Assays Utilizzando iperfosforilata Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

Metodi per la pelle Ferimento e saggi per risposte ferita<em&gt; C. elegans</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La quantificazione di adesività delle cellule endoteliali<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Un sistema semplificato per la valutazione delle cellule Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Una camera per la migrazione delle cellule di misura personalizzabile

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...