Name: in vitro cell migration and invasion assays
Uploaded: 2014-06-01T13:00:00
Description: Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

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. Cellekultur sårlukning Assay <ol><li> Frigør cellerne fra vævskulturplade anvendelse af 0,25% trypsin-EDTA-opløsning. Pellet-celler i et 15 ml konisk rør ved centrifugering aspireres supernatanten og resuspender cellerne i dyrkningsmediet. Plate et passende antal celler i en 6-brønds plade til 100% konfluens i 24 timer. Bemærk: Prøver kan være nødvendige for at bestemme tid og antallet af celler for at opnå 100% konfluens på grund af celletypen og størrelsen af brønden anvendes (for eksempel, e...

<ol>
	<li>Castellone, R. D., Leffler, N. R., Dong, L., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+tumor+cell+migration+and+metastasis+by+the+proton-sensing+GPR4+receptor.">Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor.</a> Cancer Lett. 312 (2), 197-208 (2011).</li><li>Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytoskeleton+and+cancer.">The cytoskeleton and cancer.</a> Cancer Metastasis Rev. 28, (2009).</li><li>Lauffenburger, D. A., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>Peter, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+to+apoptotic+&amp;#34;find-me&amp;#34;+signals+is+mediated+via+the+phagocyte+receptor+G2A.">Migration to apoptotic &amp;#34;find-me&amp;#34; signals is mediated via the phagocyte receptor G2A.</a> J. Biol. Chem. 283 (9), 5296-5305 (2008).</li><li>Radu, C. G., Yang, L. V., Riedinger, M., Au, M., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+chemotaxis+to+lysophosphatidylcholine+through+the+G2A+receptor.">T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.</a> Proc. Natl. Acad. Sci. U. S. A. 101 (1), 245-250 (2004).</li><li>Buul, J. D., Hordijk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+in+leukocyte+transendothelial+migration.">Signaling in leukocyte transendothelial migration.</a> Arterioscler Thromb. Vasc. Biol. (5), 824-833 (2004).</li><li>Yang, L. V., Heng, H. H., Wan, J., Southwood, C. M., Gow, A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+promoters+and+polyadenylation+regulate+tissue-specific+expression+of+Hemogen+isoforms+during+hematopoiesis+and+spermatogenesis.">Alternative promoters and polyadenylation regulate tissue-specific expression of Hemogen isoforms during hematopoiesis and spermatogenesis.</a> 228 (4), 606-616 (2003).</li><li>Yang, L. V., Radu, C. G., Wang, L., Riedinger, M., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gi-independent+macrophage+chemotaxis+to+lysophosphatidylcholine+via+the+immunoregulatory+GPCR+G2A.">Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.</a> Blood. 105 (3), 1127-1134 (2005).</li><li>Yoshida, M., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chemotaxis+and+regulation+of+flagellar+movement+by+Ca2+.">Sperm chemotaxis and regulation of flagellar movement by Ca2+.</a> Mol. Hum. Reprod. 17 (8), 457-465 (2011).</li><li>Albini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+and+endothelial+cell+invasion+of+basement+membranes.+The+matrigel+chemoinvasion+assay+as+a+tool+for+dissecting+molecular+mechanisms.">Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms.</a> Pathol. Oncol. Res. 4 (3), 230-241 (1998).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+determinants+of+metastasis.">Critical determinants of metastasis.</a> Semin. Cancer Biol. 12 (2), 89-96 (2002).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+mechanistic+insights+and+clinical+challenges.">Tumor metastasis: mechanistic insights and clinical challenges.</a> Nat. Med. 12 (8), 895-904 (2006).</li><li>Roussos, E. T., Condeelis, J. S., Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+cancer.">Chemotaxis in cancer.</a> Nat. Rev. Cancer. 11 (8), 573-587 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+3D+morphology+and+functions+on+genetically+engineered+mouse+melanoma+cells.">Comparative study of 3D morphology and functions on genetically engineered mouse melanoma cells.</a> Integr. Biol. (Camb. 4 (11), 1428-1436 (2012).</li><li>Junger, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+regulation+of+neutrophil+chemotaxis.">Purinergic regulation of neutrophil chemotaxis.</a> Cell. Mol. Life Sci. 65 (16), 2528-2540 (2008).</li><li>Lazennec, G., Richmond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+chemokine+receptors:+new+insights+into+cancer-related+inflammation.">Chemokines and chemokine receptors: new insights into cancer-related inflammation.</a> Trends Mol. Med. 16 (3), 133-144 (2010).</li><li>Huang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J. Biomed. Sci. 17, (2010).</li><li>Cortesio, C. L., Boateng, L. R., Piazza, T. M., Bennin, D. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-mediated+proteolysis+of+paxillin+negatively+regulates+focal+adhesion+dynamics+and+cell+migration.">Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration.</a> J. Biol. Chem. 286 (12), 9998-10006 (2011).</li><li>Wehrle-Haller, B., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+microtubules+and+focal+adhesion+dynamics+during+cell+migration.">Actin microtubules and focal adhesion dynamics during cell migration.</a> Int. J. Biochem. Cell Biol. 35 (1), 39-50 (2003).</li></ol>

Cell migration er et vigtigt aspekt at studere i kræftforskning, og det kan også anvendes til udviklingsmæssige, immunologiske og sårheling studier. Cellekulturen sårlukning assay og transwell celle migration og invasion-assays afslører detaljerede oplysninger om celle vandrende adfærd og kan anvendes til at undersøge de molekylære mekanismer i cellemigrering 1,2,10,14. Vores undersøgelse brugt disse celle motilitet assays til at bestemme migrationen hastigheds-og invasion kapaciteter en B16F10 mela...

Motilitet er et væsentligt element i levende celler. Cell migration er involveret i udformningen af livet, fosterudvikling, immunrespons, og mange patologiske processer, såsom cancer metastaser og inflammation 1-9. Derfor metoder til at studere celle vandringsadfærd er meget nyttige forskning værktøjer til en bred vifte af discipliner i biomedicinske videnskaber, biologi, bioteknik og felter relateret. Studiet af celle migration i kræftforskning er af særlig interesse som den vigtigste dødsårsag i kræftpatienter er relateret til metastatisk progression. For kræft til at sprede og udbrede i hele kroppen, skal kræftceller migrere og invadere gennem ekstracellulær matrix (ECM), intravasate i blodcirkulationen, tillægger et fjernt sted, og endelig ekstravasere at danne fjernt foci 1,10-12. Forskellige biologiske metoder kan anvendes til at studere disse begivenheder i detaljer. Cellekultur sår lukning en Dend den transwell migration og invasion assays er meget udbredt i det videnskabelige samfund 1,10. Disse tests kan give den nødvendige data, der kan give mulighed for en forståelse af, hvor godt en bestemt celletype kan spontant migrere eller svare på en kemo-lokkemidler og retningsbestemt migrere mod det. Adskillige vandrende fænotyper er blevet beskrevet. Celler kan migrere i en enkelt celle form, som det ses i mesenchymale eller amoeboid-lignende bevægelse eller flercellede bevægelse mærket kollektiv migration eller celle streaming 13. Fremgangsmåden til bevægelse anvendes i motile celler kan let observeres ved hjælp af cellekultur sårlukning assay. Blandt de mange måder at studere cellemigrering cellen sårlukning assayet er en af ​​de enkleste. Denne metode er nyttig til at bestemme migrationen evne helcelle masserne. Når taget et skridt videre den kan bruges til at observere de enkelte celles morfologiske kendetegn ved migration 14. Efter analysen af ​​sårlukning mange fænotyper kan blive afsløret. Måling af lukkede distance over tid, når man sammenligner med en kontrol kan afsløre specifikke ændringer migration eller en forringet vandrende fænotype, der var ukendt tidligere. Desuden kan enkelt celle lamellipodium dannelse, hale tilbagetrækning, og retningsbestemt bevægelse give fingerpeg til, hvad der kan blive forringet eller forøget i cellerne af interesse 14. The transwell migration og invasion-assays kan anvendes til at analysere evnen af enkelte celler til retningsbestemt reagerer på forskellige kemo-tiltrækningsmidler om de er kemokiner, vækstfaktorer, lipider eller nukleotider 4,5,8,15,16. Det kan også vurdere forskellen vandrende evne på grund af overekspression af en receptor 1,14. Disse assays kan også anvendes til at identificere og karakterisere de vigtige regulatorer af cellemigrering, såsom Rho familie af små GTPaser 2. Efter disse korte og let tilgængeligerende prøver, den tilstand af cellemigrering og en celles evne til at invadere i en 3-D matrix kan også bestemmes.

<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.

Sårlukkesystemet assay og transwell cellemigrering assay præsenteres her blev udført ved hjælp af musen B16F10 melanom celler som modelsystem. I sårlukning assayet blev B16F10-celler podet i en 6-brønds vævskulturplade og dyrket til 100% konfluens i 24 timer. Et sår på ca 700 um bred blev genereret ved hjælp af en pipette spids og sårlukning (celle migration) blev optaget med time-lapse mikroskopi. Alternativt kan sårlukning også blive undersøgt ved at tage snapshot billeder på forskellige tidspunkter, hv...

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

In vitro Cell Migration and Invasion Assays

Almindeligt anvendte, er meget tilgængelige metoder til at undersøge celle migration og invasion in vitro beskrevet. Den første metode er den celle sårlukning assay, der måler cellemotilitet. Den anden metode er den transwell migration og invasion assay, der vurderer det kemotaktiske og invasive kapacitet af celler.

In vitro Cell Migration og invasion Analyser

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

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.

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

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.

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.

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.

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.

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 Aggregation assays under anvendelse af hyperphosphoryleret 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.

Metoder til Hud sårbarhed og Analyser for Wound Svar i<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.

Kvantificering af endotelcelle Klæbeevne<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.

En forenklet system for Evaluering Cell Mechanosensing og 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.

En tilpasses Chamber til måling 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 ...