ECIS Connection - December 2011

The ECIS Connection - December 2011

Dr. Seema Sehrawat - ECIS Early Career Grant Recipient

Applied BioPhysics is proud to introduce Dr. Seema Sehrawat, Research Fellow at the Brigham and Women's Hospital, Harvard Medical School, as the latest recipient of the Early Career ECIS Grant Program. This program fosters the prospects of young scientists to secure initial NIH funding for health related research.

Dr. Sehrawat is testing the hypothesis that viral and bacterial pathogens regulate PKA activity in a cAMP-dependent manner to regulate specific proteins/protein complexes. She will characterize a potentially novel protein complex containing AKAP9/Epac1 at the centrosome and its role in endothelial barrier function particularly in the context of viral and bacterial infection. Using the ECIS Z-Theta she will monitor endothelial barrier function and associated cell morphology changes on a real-time basis, to elucidate the signaling mechanisms involved.

Applied BioPhysics accepts applications for the Early Career ECIS Grant on an ongoing basis. The grant provides support for researchers in the pursuit of preliminary data for their first R01. Applications should be submitted 4 months prior to the intended NIH submission date. Scientists applying for initial NSF funding are also encouraged to apply. Foreign scientists applying for initial funding from their own governments should email a letter of inquiry describing their status to Dr. Christian Renken at renken@biophysics.com for clarification of eligibility.

Radiation Research Podcast with Erik Young of Columbia University

The October 2011 edition of the Radiation Research Podcast features an interview with Erik Young, hosted by Marjan Boerma. Columbia University researchers Erik Young and Lubomir Smilenov are co-authors of the paper entitled Impedance-Based Surveillance of Transient Permeability Changes in Coronary Endothelial Monolayers after Exposure to Ionizing Radiation, appearing in the October issue of Radiation Research. A link to the podcast is provided below:

Erik Young Radiation Research Podcast

Erik Young and Lubomir Smilenov, Columbia University.

Applied BioPhysics sponsors the 1st International Meeting on Ion Channel Signaling Mechanisms

ICSM2011 took place in Marrakesh, Morocco October 31 - November 4. The meeting was organized by Dr.

Mohamed Trebak of Albany Medical College, an ECIS user studying the regulation of endothelial permeability and angiogenesis by calcium.

Mohamed Blows The Dinner Horn
Photograph courtesy of Barbara Putney

While the highlight of the meeting was the gala dinner complete with Moroccan musicians and dancers, Applied BioPhysics took special pride in the presentation given by Dr. Mohamed Trebak. His work with Dr. Arte Shinde illustrates a novel role of STIM1 in regulating vascular permeability in response to thrombin, which is completely independent of its known role as a calcium channel regulator. ECIS monitoring of barrier function in micro-dermal vascular cell and human umbilical vascular endothelial cells was the major assay used in this study (see figure below). Applied BioPhysics congratulates both Mohamed and Arte on their findings.

Thrombin (100nM) caused a decrease in TER of HUVECs (blue trace) and this decrease was inhibited in cells transfected with siRNA against STIM1 (red trace) which was rescued upon transfection into cells of an siRNA-resistant STIM1 cDNA clone (black trace).

New Tutorial on Phase-Contrast & Fluorescence Microscopy with ECIS

We are grateful to Dr. Joachim Wegener of the University of Regensburg for preparing a tutorial on Phase-Contrast & Fluorescence Microscopy with ECIS. When cells are grown on ECIS electrodes they can be examined by both phase contrast and fluorescence microscopy. Compared to ordinary cell culture substrates there are some peculiarities arising from the gold film and the chambers of the ECIS array. This tutorial gives step by step instructions on how to carry out both of these procedures effectively.

Click here to view the tutorial: Phase-Contrast & Fluorescence Microscopy with ECIS

ECIS Software

The latest Windows version of the ECIS software is v1.2.72.0. You can find which version you are running by looking at the splash screen on start-up, or by selecting Help | About.

Software Update

If your computer is connected to the internet, you can update to the latest ECIS software by selecting Help | Check for Updates. Otherwise, you can manually download the update by pasting the following url into your browser: http://www.biophysics.com/software/ECIS_Software_Update.msi

New System Install

If you wish to install the ECIS software on a separate computer for off-line analysis, you first need to download the following file (180Mb), unzip it and run 'Install.bat': by pasting the following url into your browser: http://www.biophysics.com/software/ECIS_Web_Install.zip

The unzip password is 'NewECISSoftware'. You should then update your ECIS software to the latest version as described above.

USB Driver Install

If you need to install the software on a new system for acquiring data, follow the instructions above and then install the USB driver. The latest Edgeport driver is v5.30 and is included with with the software. It can be installed by selecting Help | Edgeport USB | Install Driver, then extract the files to 'C:\EdgePort Drivers'. After plugging in the USB cable, use the 'New Hardware Wizard' to manually select the driver.

Windows 7 users in particular will need to use the Edgeport software to verify that the COM ports are numbered sequentially. Select Help | Edgeport USB | Configuration Utility.

Mac Systems

If you are using a Mac system please contact us for upgrade instructions.

Updated Manual

Please note that the manual has recently been updated and is available as a PDF from Help | Manual.

Software Features

Default Experiment Type

If you typically collect data with multiple frequencies, you can set this as the default experiment type by selecting Default Expt. Type from the Acquire menu, and then select MFT. The software will then start with that mode selected. You can also choose SFT or RTC if desired.

Set Scan Frequencies

You can manually specify the frequencies to be applied during a MFT using the Acquire | Set Scan Frequencies | Manual option. Frequencies are normally separated by a factor of 2 or 4 for analysis on a logarithmic scale. There is also a predefined option for '4/16/64 kHz' which gives a quick 3 frequency scan for cell monitoring, but is not suitable for modeling.

ECIS Webinar Schedule 2012

ECIS application webinars review the topics listed below in 20 to 30 minute, web-based, interactive seminars presented by Applied BioPhysics president and co-founder, Dr. Charles Keese.

All webinars are held at 11:00am EST. To register for a webinar, please go to:

https://appliedbiophysics.webex.com and scroll to the webinar date of interest.

ECIS Theory - 11:00 AM EST

January 10, 2012

Cell Invasion / Extravasation Assays - 11:00 AM EST

January 24, 2012

Automated Cell Migration - 11:00 AM EST

February 7, 2012

Barrier Function Assays - 11:00 AM EST

February 21, 2012

Real-time Electroporation and Monitoring - 11:00 AM EST

March 6, 2012

Cell Attachment and Spreading Measurements - 11:00 AM EST

March 20, 2012

For a more detailed description of each webinar, please visit: http://www.biophysics.com/webinar.php

New Publications

Impedance-based surveillance of transient permeability changes in coronary endothelial monolayers after exposure to ionizing radiation. EF Young and LB Smilenov. Radiat Res. 176: 415. (2011)

R-Ras interacts with filamin a to maintain endothelial barrier function. Griffiths GS, Grundl M, Allen JS 3rd, Matter ML. J Cell Physiol. Sep; 226(9):2287-96. doi: 10.1002/jcp.22565. (2011)

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via the activation of endothelial nitric oxide synthase. Bevan HS, Slater SC, Clarke H, Cahill PA, Mathieson PW, Welsh GI, Satchell SC. Am J Physiol Renal Physiol. Jul 20 (2011)

Whole cell based electrical impedance sensing approach for rapid nanotoxicity assay, E. Hondroulis, C. Liu, C.-Z. Li, Nanotechnology, 31, 315103. (2010)

Endothelial Reticulon-4B (Nogo-B) regulates ICAM-1-mediated leukocyte transmigration and acute inflammation. Di Lorenzo A., Manes T.D., Wright P.L. and Sessa W.C. Blood. 117(7):2284-95. (2011)

CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations. Zheng X., Xu C., Di Lorenzo A., Kleaveland B., Zou Z., Seiler C., Chen M., Cheng L., Xiao J., He J., Pack M.A., Sessa W.C., Kahn M.L. JCI. 120(8):2795-804. (2011)

Septin-2 Mediates Airway Epithelial Barrier Function in Physiologic and Pathologic Conditions
Venkataramana K. Sidhaye, Eric Chau, Patrick N. Breysse, and Landon S. King. Am. J. Respir. Cell Mol. Biol. 45:120-126. (2011)

Impedance analysis of adherent cells after in situ electroporation: Non-invasive monitoring during intracellular manipulations. JA Stolwijk, C Hartmann, P Balani, S Albermann, CR Keese, I Giaever, and J Wegener Biosens Bioelectron. (2011)

Site-Directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade. Gemma E. White, Eileen McNeill, Ivy Christou, Keith M. Channon, and David R. Greaves. Mol. Pharmacol. 80:328-336. (2011)

Effects of osmolarity on human epithelial conjunctival cells using an electrical technique. M Bellotti, W Bast, A Berra, and FJ Bonetto. Graefes Arch Clin Exp Ophthalmol. (2011)

Dynamic Changes of Acoustic Load and Complex Impedance as Reporters for Cytotoxicity of Small Molecule Inhibitors. A Janshoff, M Tarantola, E Sunnick, S David, AK Marel, and A Kunze. Chem Res Toxicol. (2011)

R-Ras interacts with filamin a to maintain endothelial barrier function. Griffiths GS, Grundl M, Allen JS 3rd, Matter ML. J Cell Physiol. Sep;226(9):2287-96. doi: 10.1002/jcp.22565. (2011)

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species.
Youxue Wang, Qun S. Zang, Zijuan Liu, Qian Wu, David Maass, Genevieve Dulan, Philip W. Shaul, Lisa Melito, Doug E. Frantz, Jessica A. Kilgore, Noelle S. Williams, Lance S. Terada, and Fiemu E. Nwariaku
Am J Physiol Cell Physiol. 301:C695-C704. (2011)

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase. Heather S. Bevan, Sadie C. Slater, Hayley Clarke, Paul A. Cahill, Peter W. Mathieson, Gavin I. Welsh, and Simon C. Satchell. Am J Physiol Renal Physiol. 301:F733-F742. (2011)

Metastasis Suppressor-1, MTSS1, Acts as a Putative Tumour Suppressor in Human Bladder Cancer
PENG DU, LIN YE, FIONA RUGE, YONG YANG, and WEN G. JIANG. Anticancer Res. 31:3205-3212. (2011)

Adenosine monophosphate-activated kinase α1 promotes endothelial barrier repair. Judy Creighton, MingYuan Jian, Sarah Sayner, Mikhail Alexeyev, and Paul A. Insel. FASEB J. 25:3356-3365. (2011)

Endogeneity between internationalization and knowledge creation of global R&D leader firms: an econometric approach using Scoreboard data. Areti Gkypali, Kostas Tsekouras, and Nick von Tunzelmann. Ind. Corp. Change. published 29 September 10.1093/icc/dtr057. (2011)

Nuclear Factor of Activated T Cells 5 Regulates Vascular Smooth Muscle Cell Phenotypic Modulation
Julia A. Halterman, H. Moo Kwon, Ramin Zargham, Pamela D. Schoppee Bortz, and Brian R. Wamhoff
Arterioscler Thromb Vasc Biol. 31:2287-2296. (2011)

Differential Regulation of Endothelial Cell Permeability by High and low Doses of OxPAPC
Vitaliy Starosta, Tinghuai Wu, Alejandro Zimman, Donald Pham, Xinyong Tian, Olga Oskolkova, Valery Bochkov, Judith A Berliner, Anna A Birukova, and Konstantin G Birukov. Am. J. Respir. Cell Mol. Biol. published 13 October 10.1165/rcmb.2011-0153OC (2011)

Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties. Ramadan M. Sarrab, Rachel Lennon, Lan Ni, Matthew D. Wherlock, Gavin I. Welsh, and Moin A. Saleem. Am J Physiol Renal Physiol. 301:F1131-F1138. (2011)

The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading
Gary J. Doherty, Monika K. Åhlund, Mark T. Howes, Björn Morén, Robert G. Parton, Harvey T. McMahon, and Richard Lundmark. Mol. Biol. Cell. 22:4380-4389. (2011)

Pericyte-Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier. Paul G. McGuire, Sampathkumar Rangasamy, Joann Maestas, and Arup Das Arterioscler Thromb Vasc Biol. 31:e107-e115. (2011)

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Have you recently published an article that includes the use of ECIS? If so, submit your publications to Applied BioPhysics via email to Nancy Vlahos at vlahos@biophysics.com. We will announce your article in our newsletter, post it on our website and send you 2 FREE 8 well arrays!

Upcoming Events

Representatives from Applied BioPhysics will be at the following tradeshows and events:

Intestinal Pathobiology Symposia
March 1 - 2
Georgia State University
Atlanta, GA
dmerlin@gsu.edu

Society of Toxicology
March 12 - 14
San Francisco, CA

American Association for Cancer Research
March 31-April 4
Chicago, IL

Tip of the Month:

Don't Forget Capacitance

Capacitance at high frequency can be used to determine the percentage of substrate covered with cells - a very useful quantity measured using ECIS without opening the incubator door.

Most papers presenting ECIS data show resistance (R) and sometimes impedance (Z), but rarely present capacitance (C) data. The likely reason is that in the intermediate AC frequency range where most ECIS measurements are made, the capacitance undergoes relatively small changes. This is not true, however, at high frequencies (32,000 Hz and higher). Under high frequency conditions there is a noticeable drop in the measured capacitance that is directly related to cell coverage.

Consider the capacitance of 10E+ electrodes at 40,000 Hz. Without cells, the capacitance of a well is ~ 60 nanofarads. As cells attach and spread upon the electrodes the capacitance drops to less than 10 nanofarads. The relationship between capacitance measured at high frequency and the amount of cell-free gold open to solution is, for most cells, a linear one. In other words, the capacitance at high frequency is a direct measure of how much of the substrate is cell-free.

In the situation described above, when the capacitance of a partially cell-covered electrode is 30 nanofarads, the amount of cell-free substrate is calculated as below:

Percent of cell-free substrate = 100% (Cmeasured - Cconfluent)/(Ccell free - Cconfluent)

Percent of cell-free substrate = 100% (30-10)/(60-10) = 100% (20/50) = 40%

or 60% cell coverage.

Knowing the degree of cell coverage is useful when doing wound healing assays and is especially important when modeling cell data. The ECIS model requires a confluent cell layer to be valid, and this can be quickly verified by checking the value of the high frequency capacitance.

ECIS Humor

Need a good laugh? Visit the ECIS Cartoons page of our website to view cartoons by Catherine, our in-house cartoonist, to start your day with a smile.

Are you the creative type? Submit one of your own cartoons; if we post it on our website we will send you a free array!