Name: a quantitative evaluation of cell migration by the phagokinetic track motility assay
Uploaded: 2012-12-04T12:00:00
Description: Watch this Scientific Journal Video about A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay at JoVE.com

This work was supported by grants from the National Institutes of Health (AI050677, HD-051998, and GM103433), a Malcolm Feist cardiovascular research fellowship, and an American Heart Association predoctoral fellowship (10PRE4200007).

1. Preparation of Gelatin-coated Coverslips
<ol>
	<li>Place acid-washed glass coverslips (15 mm in diameter) in a sterile plastic 100 mm dish(es). Place 8-9 coverslips per dish and make sure they are not touching each other or the sides of the dish..</li>
</ol>
Note: Coverslips, needles and tweezers need to be sterile to eliminate possible contaminating microorganisms, as well as endotoxins that will affect cellular functions, including motility.
<ol start="2">
	<li>Weigh th...

<ol>
	<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+cell+motility+during+embryogenesis.">The control of cell motility during embryogenesis.</a> Cancer Metastasis Rev. 4, 59-79 (1985).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+and+go+traffic+to+tune+T+cell+responses.">Stop and go traffic to tune T cell responses.</a> Immunity. 21, 305-314 (2004).</li><li>Mutsaers, S. E., Bishop, J. E., McGrouther, G., Laurent, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tissue+repair:+from+wound+healing+to+fibrosis.">Mechanisms of tissue repair: from wound healing to fibrosis.</a> Int. J. Biochem. Cell Biol. 29, 5-17 (1997).</li><li>Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+proteins+in+migration+and+invasion.">Polarity proteins in migration and invasion.</a> Oncogene. 27, 6970-6980 (2008).</li><li>Parsons, J. T., Horwitz, A. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion:+integrating+cytoskeletal+dynamics+and+cellular+tension.">Cell adhesion: integrating cytoskeletal dynamics and cellular tension.</a> Nat. Rev. Mol. Cell Biol. 11, 633-643 (2010).</li><li>Mitchison, T. J., Cramer, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-based+cell+motility+and+cell+locomotion.">Actin-based cell motility and cell locomotion.</a> Cell. 84, 371-379 (1996).</li><li>Pollard, T. D., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+motility+driven+by+assembly+and+disassembly+of+actin+filaments.">Cellular motility driven by assembly and disassembly of actin filaments.</a> Cell. 112, 453-465 (2003).</li><li>Bretscher, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+membrane+flow+and+the+cytoskeleton+to+cooperate+in+moving+cells.">Getting membrane flow and the cytoskeleton to cooperate in moving cells.</a> Cell. 87, 601-606 (1996).</li><li>Albrecht-Buehler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phagokinetic+tracks+of+3T3+cells.">The phagokinetic tracks of 3T3 cells.</a> Cell. 11, 395-404 (1977).</li><li>Smith, M. S., Bentz, G. L., Alexander, J. S., Yurochko, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cytomegalovirus+induces+monocyte+differentiation+and+migration+as+a+strategy+for+dissemination+and+persistence.">Human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence.</a> J. Virol. 78, 4444-4453 (2004).</li><li>Palmisano, R., Itoh, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Metalloproteinase+Protocols.">Matrix Metalloproteinase Protocols.</a> Methods in Molecular Biology. 622, 379-392 (2010).</li><li>Ohta, H., 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=HOXD3-Overexpression+Increases+Integrin+Alpha+V+Beta+3+Expression+and+Deprives+E-Cadherin+while+It+Enhances+Cell+Motility+in+A549+Cells.">HOXD3-Overexpression Increases Integrin Alpha V Beta 3 Expression and Deprives E-Cadherin while It Enhances Cell Motility in A549 Cells.</a> Clinical and Experimental Metastasis. 7-8, 381-390 (2006).</li><li>Kawa, S., Kimura, S., Hakomori, S., Igarashi, Y. <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+chemotactic+motility+and+trans-endothelial+migration+of+human+neutrophils+by+sphingosine+1-phosphate.">Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate.</a> FEBS Lett. 420, 196-200 (1997).</li><li>Kawamura, K., 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=N-WASP+and+WAVE2+acting+downstream+of+phosphatidylinositol+3-kinase+are+required+for+myogenic+cell+migration+induced+by+hepatocyte+growth+factor.">N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.</a> J. Biol. Chem. 279, 54862-54871 (2004).</li><li>Ando, Y., Jensen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+insulin-like+growth+factor+I+enhance+keratinocyte+migration.">Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration.</a> J. Invest. Dermatol. 100, 633-639 (1993).</li><li>Todt, J. 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=Effects+of+tumor+necrosis+factor-alpha+on+human+trophoblast+cell+adhesion+and+motility.">Effects of tumor necrosis factor-alpha on human trophoblast cell adhesion and motility.</a> Am. J. Reprod. Immunol. 36, 65-71 (1996).</li><li>McAuslan, B. R., Reilly, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+phagokinesis+in+response+to+specific+metal+ions.">Endothelial cell phagokinesis in response to specific metal ions.</a> Exp. Cell. Res. 130, 147-157 (1980).</li><li>Smith, M. S., Bentz, G. L., Smith, P. M., Bivins, E. R., Yurochko, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCMV+activates+PI(3)K+in+monocytes+and+promotes+monocyte+motility+and+transendothelial+migration+in+a+PI(3)K-dependent+manner.">HCMV activates PI(3)K in monocytes and promotes monocyte motility and transendothelial migration in a PI(3)K-dependent manner.</a> J. Leukoc. Biol. 76 (3), 65-76 (2004).</li><li>Smith, M. S., 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=Roles+of+phosphatidylinositol+3-kinase+and+NF-kappaB+in+human+cytomegalovirus-mediated+monocyte+diapedesis+and+adhesion:+strategy+for+viral+persistence.">Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence.</a> J. Virol. 81, 7683-7694 (2007).</li><li>Bentz, G. L., Yurochko, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CMV+infection+of+endothelial+cells+induces+an+angiogenic+response+through+viral+binding+to+EGF+receptor+and+beta1+and+beta3+integrins.">Human CMV infection of endothelial cells induces an angiogenic response through viral binding to EGF receptor and beta1 and beta3 integrins.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 5531-5536 (2008).</li><li>Chan, G., Nogalski, M. T., Yurochko, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+EGFR+on+monocytes+is+required+for+human+cytomegalovirus+entry+and+mediates+cellular+motility.">Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 22369-22374 (2009).</li><li>Nogalski, M. T., Chan, G., Stevenson, E. V., Gray, S., Yurochko, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCMV-Regulated+Paxillin+in+Monocytes+Links+Cellular+Pathogenic+Motility+to+the+Process+of+Viral+Entry.">HCMV-Regulated Paxillin in Monocytes Links Cellular Pathogenic Motility to the Process of Viral Entry.</a> J. Virol. 85, 1360-1369 (2011).</li><li>Turkevich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+nucleation+and+growth+processes+in+the+synthesis+of+colloidal+gold.">A study of the nucleation and growth processes in the synthesis of colloidal gold.</a> Discuss. Faraday. Soc. 11, 55-75 (1951).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size+and+sol+stability+in+mental+colloids.">Particle size and sol stability in mental colloids.</a> Colloid &#38; Polymer Science. 250, 736-741 (1972).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+nucleation+for+the+regulation+of+the+particle+size+in+monodisperse+gold+suspensions.">Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions.</a> Phys. Sci. 241, 20-22 (1973).</li><li>Zetter, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+capillary+endothelial+cell+migration.">Assay of capillary endothelial cell migration.</a> Methods Enzymol. 147, 135-144 (1987).</li><li>Scott, W. N., McCool, K., Nelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+the+production+of+gold+colloid+monolayers+for+use+in+the+phagokinetic+track+assay+for+cell+motility.">Improved method for the production of gold colloid monolayers for use in the phagokinetic track assay for cell motility.</a> Anal. Biochem. 287, 343-344 (2000).</li><li>Wang, S. Y., Mak, K. L., Chen, L. Y., Chou, M. P., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+blood+monocyte:+two+subpopulations+with+different+sizes,+phenotypes+and+functions.">Heterogeneity of human blood monocyte: two subpopulations with different sizes, phenotypes and functions.</a> Immunology. 77, 298-303 (1992).</li><li>Windler-Hart, S. L., Chen, K. Y., Chenn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+behavior+screen:+identification,+sorting,+and+enrichment+of+cells+based+on+motility.">A cell behavior screen: identification, sorting, and enrichment of cells based on motility.</a> BMC Cell Biol. 6, 14 (2005).</li><li>Pan, 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=Size-dependent+cytotoxicity+of+gold+nanoparticles.">Size-dependent cytotoxicity of gold nanoparticles.</a> Small. 3, 1941-1949 (2007).</li><li>Rodriguez, L. G., Wu, X., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+assay.">Wound-healing assay.</a> Methods Mol. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+suppressor+DAPK+inhibits+cell+motility+by+blocking+the+integrin-mediated+polarity+pathway.">The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway.</a> J. Cell Biol. 172, 619-631 (2006).</li><li>Benazeraf, B., 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=A+random+cell+motility+gradient+downstream+of+FGF+controls+elongation+of+an+amniote+embryo.">A random cell motility gradient downstream of FGF controls elongation of an amniote embryo.</a> Nature. 466, 248-252 (2010).</li></ol>

The phagokinetic track motility assay presented in this article is a simple and highly effective method for quantitative analysis of cell migration. Because multiple cell types can be analyzed9-17, this method has the potential broad usage across multiple disciplines. The use of colloidal gold-coated glass coverslips allows for the measurement of a track area cleared by a moving cell. The assay can measure the effect of different stimuli (i.e. growth factors, purified ECM ligands, viruses, bac...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Glass Coverslips (15 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-83</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin 300 Bloom</td>
			<td>Sigma-Aldrich</td>
			<td>G-1890</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tetrachloroauric Acid Trihydrate</td>
			<td>Fisher Chemical</td>
			<td>G54-1</td>
			<td>14.5 mM (a final working solution)</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>BP327-500</td>
			<td>0.5% (a final working solution)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>O4042</td>
			<td>3% (a final working solution)</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Dish</td>
			<td>Sarstedt</td>
			<td>83.1802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>24-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-84</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Techne Oven Hybridiser HB-1D</td>
			<td>LabPlanet</td>
			<td>2040500</td>
			<td>The standard laboratory oven will suffice</td>
		</tr>
		<tr>
			<td>10 ml Serological Pipettes</td>
			<td>Sarstedt</td>
			<td>86.1254.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipet-Aid Filler/Dispenser</td>
			<td>Drummond</td>
			<td>13-681-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>P200 Single-Channel Manual Pipette</td>
			<td>Rainin</td>
			<td>PR-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 ml Barrier Tips</td>
			<td>CLP</td>
			<td>BT200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td><a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a></td>
			<td>&nbsp;</td>
			<td>License: Public Domain</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE300 with a photometrics CoolSNAPfx monochrome 12-bit CCD camera</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>Discontinued; The most comparable specification has Nikon Eclipse Ti, but a lower end Nikon 80i will be suitable as well. Other brands also provide comparable microscopes.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Note: The reagents and equipment listed below have been utilized by us in our various studies. Other supplies, suppliers, reagents, and equipment can be used, as long as they have similar specifications.</td>
		</tr>
	</tbody>
</table>

a quantitative evaluation of cell migration by the phagokinetic track motility assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

Shown is an example of pictures taken under a light microscope showing a track area cleared by a single cell (a monocyte from our experiments is shown in Figure 2). Non-motile cells create characteristic small, oval or circle-shaped tracts around themselves indicating a low basal level of movement for these unstimulated cells (Figures 2A and 2B). In contrast, highly motile cells [in our system, human cytomegalovirus (HCMV)-infected cells] are characterized by a directional movement shown...

Watch this Scientific Journal Video about A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay at JoVE.com

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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Research

JoVE Journal

Immunology and Infection

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...