Automated Cell Migration Assay


The ECIS Method

Cell Migration Measurements

How does it work?

Traditional Scratch Method

Scratch Method V. ECIS Wound Healing Assay

Electric Fence Cell Migration Assay


Jove Video: Measurement of Cellular Chemotaxis with ECIS\Taxis (11:32)

ECIS Wounding Assay

The ECIS Wound Healing Assay replaces the traditional "scratch" or "scrape" assay. Instead of disrupting the cell layer mechanically with a toothpick, needle or pipette tip and following the migration of cells to "heal" the wound with a microscope, we employ electric signals to both wound and monitor the healing process.

ECIS electrical wounding is only directed at the small population of cells in contact with the active 250 micrometer diameter ECIS electrode, producing a well defined 250 micrometer wound that can be verified both with the ECIS measurement and with vital staining.

Unlike the traditional scrape method, with the ECIS Wound your protein coating is unaffected by the current, it remains fully intact.

 

ECIS Cell Migration Measurements

Once ECIS electrically wounds the cells, it returns to its normal mode to immediately follow the healthy neighboring cells as they migrate inward to replace the killed cells. Traditionally, these measurements are carried out with extensive labor including microscopy and researcher quantification. The ECIS Wound-Healing/Cell Migration Assay is a completely automated assay requiring a minimum of labor. Both cell wounding and measurements of the subsequent healing process are carried out under computer control without opening the door of the incubator.
How does it work?

ECIS has now been modified (Patent pending) such that automated assays of this sort can be performed. In normal ECIS measurements, a current of less than a microampere is normally used. This is undetected by the cells, and, in its measuring mode, ECIS essentially eavesdrops on cell behavior electrically. When the current is boosted 1000 fold to a milliampere, the resulting voltages across the cell membranes result in electroporation. If this is applied for only a few milliseconds, the cells recover and it is possible to insert impermeable molecules including DNA constructs into the cytoplasm. When the high current is applied for several seconds, cell death ensues due to severe electroporation and possible local heating effects.

The ECIS wound is very well defined, as it includes only those cells on the 250 Ám diameter electrode. Death can be verified both with the ECIS measurement and with vital staining.

Typical ECIS data involving this assay is shown in the figure below. Here BSC1 cells were first grown as complete monolayers and the impedance traces from four confluent wells can be seen on the graph. At the arrow, an elevated field was applied to two of the wells, wounding the cells on the small electrode and causing the impedance to drop to that of an open electrode. Over time these two traces return to control values, as the healthy cells outside of the small electrode migrate inward to repopulate the wounded area and replace their dead cohorts (healing). These types of data are highly reproducible and respond to culture conditions.

The figure to the right shows an ECIS electrode covered with MDCK cells 24 hours after the elevated field wounding took place. Note the subtle radial patterns indicated by the arrows as the cells have migrated inward. MDCK Cells wounded with ECIS Automated Cell Wounding.

This is a completely automated assay requiring a minimum of labor. Both cell wounding and measurements of the subsequent healing process are carried out under computer control without opening the door of the incubator.

Still Starting from "Scratch"?

Wound healing assays to monitor cell migration have been carried out in tissue culture for many years to estimate the migration and proliferation rates of different cells and culture conditions. These assays generally involve first growing a confluent cell monlayer. A small area is then disrupted and a group of cells destroyed or displaced by scratching a line through the layer with an object such as toothpicks, pippette tip, rakes or needles and almost always "scrape" off the cells protein coat. The open gap is then inspected microscopically over time as the cells move in and fill the damaged area. This "healing" can take from several hours to over a day depending on the cell type, conditions and the extent of the "wounded" region.
Scratch Method V. ECIS Wound Healing Assay

Scratch Method
ECIS Wound Healing Assay
The scratch method requires hands-on measurements.
ECIS measurements are automated, quantifying data in real-time.
The scratch itself often varies and is not highly reproducible.
The ECIS Elevated Field Module produces a precise 250 micrometer wound every time.
The traditional scratch method almost always "scraped" off the cell's protein coat.
With the ECIS Wound the cell's protein coating is unaffected by the current, it is not "scraped" off.
Micrographs were taken after a traditional scratch wound assay of NRK cells 6 hours after wounding(panel I), 12 hours after wounding(panel II), and 23 hours after wounding(panel III).

Note: A confluent layer of NRK cells were scraped/wounded in a non-uniform pattern of unknown area using the tip of a pipet. The pictures show the cells as they begin to migrate toward the center of the wound. Cells must be counted and compared to establish migration rate.

Micrographs were taken from ECIS arrays before (panel I), immediately after (panel II), 2 h after (panel III), and 4 h after (panel IV) wounding (arrows in a show the corresponding time points). Note that the high electrical current completely killed endothelial cells attached to the microelectrode (panel II), and the rise of TEER was a result of the surrounding viable cells migrating into the wounded electrodes (panels III and IV). The micrographs are representative of four ECIS wells at each time point. Scale bar, 125 ÁM

Lee, JF, Zeng, Q, Ozaki, H, Wang, L, Hand, AR, Hlam T, Wang, E, Lee, MJ, "Dual Roles of Tight Junction-associated Protein, Zonula Occludens-1, in Sphingosine 1-Phosphate-mediated Endothelial Chemotaxis and Barrier Integrity," JBC: 29190-29200 (2006).


ECIS Electric Fence Cell Migration Measurements

The "Electric Fence" Cell Migration Assay

Applied Biophysics has developed a novel impedance-based technique called “The Electric Fence” to measure the rates of cell migration.

The Electric Fence differs from the Wounding Assay in that it prevents the cells from actually growing on the electrode while a confluent layer develops around the electrode . When the "electric fence" is activated, a series of high field electric pulses are applied which prevent the cells from attaching and spreading onto the measurement electrode. When the electric fence is turned off, the cells in the surrounding confluent layer migrate into the open space left by the electric fence.


electric fence graph

The progress of the migration into this open space is very precisely monitored and from this data a migration rate is calculated.  The key advantage of the Electric Fence over the Wounding Assay is the surface which the cells migrate onto has not been modified in any manner by previous cell growth, should a protein layer have been added prior to the experiment. 



Wound Healing Assay
Related ECIS Publications

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair. Katherine R. Schiller, Peter J. Maniak, and Scott M. O'Grady. Am J Physiol Cell Physiol. 2010; 299:C912-C921.

Effects of (-)-epigallocatechin gallate on RPE cell migration and adhesion. CM Chan, JH Huang, HS Chiang, WB Wu, HH Lin, JY Hong, and CF Hung. Mol Vis. 2010; 16: 586. 

Zeaxanthin inhibits PDGF-BB-induced migration in human dermal fibroblasts. NL Wu, YC Chiang, CC Huang, JY Fang, DF Chen, and CF Hung. Exp Dermatol. 2010. 

Effects of Negative Pressures on Epithelial Tight Junctions and Migration in Wound Healing. Chih-Chin Hsu, Wen Chung Tsai, Carl Pai-Chu Chen, Yun-Mei Lu, and Jong-Shyan Wang. Am J Physiol Cell Physiol. published 5 May 2010, 10.1152/ajpcell.00504.2009.

Irina Gorshkova, Donghong He, Evgeny Berdyshev, Peter Usatuyk, Michael Burns, Satish Kalari, Yutong Zhao, Srikanth Pendyala, Joe G. N. Garcia, Nigel J. Pyne, David N. Brindley,and Viswanathan Natarajan, “Protein Kinase C Regulates Sphingosine 1-Phosphate-mediated Migration of Human Lung Endothelial Cells through Activation of Phospholipase D2, Protein Kinase C, and Rac1.” Journal of Biological Chemistry (Vol. 283, No. 17, April 25, 2008). 

Jiang, W.G., Martin, T.A., Lewis-Russell, J.M., Douglas-Jones, A., Ye, L., Mansel, R.E., "Eplin-alpha expression in human breast cancer, the impact on cellular migration and clinical outcome." Molecular Cancer: 7:71 (2008).

Saxena, N.K., Sharma, D., Ding, X. Lin, S., Marra, F., Merline, D. Anania, F., "Concomitant Activation of the JAK/STAT, P13K/AKT and ERK Signaling is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells." Cancer Research: 2497-2507 (2007).

Earley, S., Plopper, G.E., "Disruption of focal adhesion slows transendothelial migration of AU-565 breast cancer cells." Biochemical and Biophysical Research Communications: 405-412 (2006).

Sapper, A., Wegener, J., Janshoff, A., "Cell motility probed by noise analysis of thickness shear mode resonators." Anal Chem. 15;78(14):5184-91, (2006).

Ren, J., Xiao, Y., Singh, L.S., Zhao, X., Zhao, Z., Feng, L., Rose, T.M., Prestwich, G.D., Xu, Y.,"Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells." Cancer Res 2006; 66: (6) (2006).

Waters, C.M., Long, J., Gorshkova, I., Fujiwara, Y., Connell, M., Belmonte, K.E., Tigyi, G., Natarajan, V., Pyne, S., Pyne, N.J., "Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1." The FASEB Journal Express Article 10.1096/fj.05-4810fje (2005).

Charrier, L., Yan, Y., Driss, A., Laboisse, C.L., Sitaraman, S.V. and Merlin, D.,"ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers." AJP Gastrointest Liver Physiol. 288:346-353, (2005).

Kucharzik, T., Lugering, A., Yan, Y., Driss, A., Charrier, L., Sitaraman, S., Merlin, D., "Activation of epithelial CD98 glycoprotein perpetuates colonic inflammantion", Laboratory Investigation 1-10 (2005)

Charrier, L., Yan, Y., Driss, A., Laboisse, C.L., Sitaraman, S.V., Merlin, D. "ADAM-15 Inhibits Wound Healing in Human Intestinal Epithelial Cell Monolayers." Am J Physiol Gastrointest Liver Physiol. [Epub ahead of print] (2004).

Keese, Charles R., Wegener, Joachim, Walker, Sarah R., and Giaever, Ivar. "Electrical Wound-healing assay for cell in vitro". PNAS 101: 1554-1559 (2004).

Hug, T.S. "Biophysical methods for monitoring cell-substrate interactions in drug discovery." Assay Drug Dev Technol. 1(3):479-88. (2003).

Lundien, M.C., Mohammed, K.A., Nasreen, N., Tepper, R.S., Hardwick, J.A., Sanders, K.L., Van Horn, R.D., Antony, V.B. "Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury." J Clin Immunol. 22(3):144-52 (2002).

Wegener, J., Keese, C.R., Giaever, I, "Recovery of Adherent Cells After In Situ Electroporation Monitored Electronically" Bio Techniques 33(2), 348ff (2002).

Hadjout, N., Laevsky, G., Knecht, D.A., Lynes ,M.A., "Automated real-time measurement of chemotactic cell motility." Biotechniques, 31 (5): 1130-1138 (2001).

Lo, C.M., Linton, M., Keese, C.R., Giaever, I. "Correlated motion and oscillation of neighboring cells in vitro." Cell Commun Adhes. 8(3):139-45 (2001).

Burns, A.R., R.A. Bowden, S.D. MacDonell, D.C. Walker, T.O. Odebunmi, E.M. Donnachie, S.I. Simon, M.L. Entman, and C.W. Smith. "Analysis of Tight Junctions During Neutrophil Transendothelial Migration." J. of Cell Sci, 113:45-75. (2000).

Huang, C.N., Lo, C.M., Hsu, T.C., Tsay, G.J. "Sera from patients with scleroderma inhibit fibroblast micromotions monitored electrically." J Rheumatol. 26(6):1312-7 (1999).

Noiri, E., Lee, E., Testa, J., Quigley, J., Colflesh, D., Keese, C., Giaever, I. and Goligorsky, M., "Podokinesis in endothelial cell migration: role of nitric oxice", Am. J. Physiol. 43, 236C (1998).

Moy A.B., Van Engelenhoven,J., Bodmer, J., Kamath, J., Keese, C.,Giaever, I., Shasby,S., and Shasby, D.M., "Histamine and Thrombin Modulate Endothelial Focal Adhesion Through Centripetal and Centrifugal Forces" J Clin. Inv. (1996).

Burns, A., Walker, D., Brown, E., Thurmon, L., Bowden, R., Keese, C., Simon, S., Entman, M.and Smith, W., "Neutrophil Transendothelial Migration Is Independent of Tight Junctions and Occurs Preferentially at Tricellular Corners", J. of Immunol. 22, 2893-2903 (1997).st. 97, 1020-1027 (1996).

Lo, C.M., Keese, C.R., Giaever, I. "Monitoring motion of confluent cells in tissue culture." Exp Cell Res. 204(1):102-9 (1993).