Automated Cell Migration Assay
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.
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.
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.
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).
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.
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).
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