Tip of the Month - ECIS® Array inoculation

Careful inoculation is the key to good reproducibility from well to well and from experiment to experiment. It is most important to obtain a uniform inoculation of the well bottom. To accomplish this, wells should receive an even "snowfall" of cells so that each electrode has approximately the same number of cells falling on its surface - this can be facilitated by attentively following the four guidelines below:

1. Make up a mono-disperse cell suspension.

For some cell lines this is easily achieved, but for others, particularly if cells have been attached and spread for long periods of time, clumping takes place and the use of EDTA followed by longer trypsinization may be needed. The goal is to achieve is a clearly mono-disperse suspension of single cells. 

2. Keep the suspension agitated

It is important to keep the cell suspension uniform so each well receives approximately the same number of cells; agitate the cell suspension frequently to prevent settling of the cells.

3. Add the suspension to an empty well

If possible, avoid mixing the cell suspension with liquid already in the well. It is best to remove all media from wells before adding the cell suspension such that no mixing within the wells is required. If a cell suspension must be added to liquid already in the well, thorough mixing of the two solutions is essential.

4. Avoid unwanted convection during cell settling and attachment

Temperature considerations are often overlooked and can be extremely important when dealing with cell distribution in small wells. If the temperature of the cell suspension is lower than that of the incubator, when the array is placed in the incubator, the wells will be heated from the bottom. This will cause a convection cell to form, where medium rises in the center and falls back down the walls of wells. Due to this flow, as cells attempt to fall to the central region of the well, they are swept upward. The overall effect is that the cell density becomes reduced in the central regions of the well. This is very undesirable especially with the 1E arrays and 10E arrays with their centrally located measuring electrodes. (see figure)

Here are two ways to help mitigate this problem:

1) Simply inoculate the ECIS arrays outside of the incubator using medium at (or warmer than) room temperature and then wait 20 to 30 minutes before placing the array in the incubator space. Since there is no heating from below, there is no thermal convection as the cells settle over the substrate and begin to attach to the surface. We recently started using this approach, and the results have been most satisfactory. CO2 dependent medium would experience some pH increase out of the incubator in this protocol, and one could consider ways to avoid this. In our hands, however, we have not found a pH drift to be a problem, as once in the incubator space, we observe normal cell attachment and spreading impedance data.

2) Alternatively, if solution #1 cannot be used, we then recommend using a cell suspension and array that are pre-warmed to incubator temperature and immediately place the array into the incubator upon inoculation. The goal again is to prevent thermal convection by having no heating of the wells taking place during cell settling.
ECIS Application Webinars

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 11:00 am EST. To register for a webinar, please go to: https://appliedbiophysics.webex.com and scroll to the webinar date of interest.  

Automated Cell Migration – November 6, 2018

Barrier Function Assays – November 20, 2018

Real-time Electroporation and Monitoring – December 4, 2018
Latest ECIS Software   
The latest version of the software is v1.2.215, which can be downloaded from Help | Check for Updates, or:

Software Tip

When you wish to copy a displayed graph for use in a report or publication, simply go to Edit | Copy Graph, to copy the current view to the clipboard, and then Paste in your new document. For more options use Edit | Export Graph, which will open the graph in a separate window. From this window you can resize the plot and save in a number of different file formats using File | Save As. If you save the plot as a Matlab Figure (*.fig), then you can reload the figure in the future if you need to change the plot size or format (use File | Open in the ECIS software). If you have access to Matlab, you can also open the figure and edit the axis labels or title.
Tradeshows & Events 

Dec 8 – 12, 2018
San Diego, CA

SOT Annual Meeting
March 10-14, 2019
Baltimore, MD

AACR Annual Meeting 2019
March 29-April 3, 2019
Atlanta, GA

ECIS School Graduates - August 2018

From left to right: Dennis Grab - Uniformed Services University, Ivar Giaever - Applied BioPhysics, Charles Keese - Applied BioPhysics, Jessica Neder - University of Maryland, Lili Gu - University of Maryland, Lin Zou - University of Maryland.
New Publications

Many new publications have been added to the Applied BioPhysics ECIS publications database.  The following are just a selection of those added.

Shamseddin, Aly et al. “Resveratrol-Linoleate Protects from Exacerbated Endothelial Permeability via a Drastic Inhibition of the MMP-9 Activity.” Bioscience Reports 38.4 (2018): BSR20171712. Web.

McRae, MaryPeace et al. “Characterization of Cell-Cell Junction Changes Associated with the Formation of a Strong Endothelial Barrier.” Tissue Barriers 6.1 (2018): e1405774. Web.

Zhang, Rongbiao et al. “A Cell Viability Assessment Method Based on Area-Normalized Impedance Spectrum (ANIS).” Biosensors and Bioelectronics 110 (2018): 193–200. Web.

Vornhagen, Jay et al. “Group B Streptococcus Exploits Vaginal Epithelial Exfoliation for Ascending Infection.” Journal of Clinical Investigation 128.5 (2018): 1985–1999. Web.

Mikó, Edit et al. “Lithocholic Acid, a Bacterial Metabolite Reduces Breast Cancer Cell Proliferation and Aggressiveness.” Biochimica et Biophysica Acta (BBA) - Bioenergetics 1859.9 (2018): 958–974. Web.

Xie, Linan et al. “Methylglucosylation of Aromatic Amino and Phenolic Moieties of Drug-like Biosynthons by Combinatorial Biosynthesis.” Proceedings of the National Academy of Sciences 115.22 (2018): E4980–E4989. Web.

Barrett, C. D. et al. “Blood Clotting and Traumatic Injury with Shock Mediates Complement-Dependent Neutrophil Priming for Extracellular ROS, ROS-Dependent Organ Injury and Coagulopathy.” Clinical & Experimental Immunology (2018): n. pag. Web.

Béguin, Eelke P. et al. “Integrated Proteomic Analysis of Tumor Necrosis Factor α and Interleukin 1β-Induced Endothelial Inflammation.” Journal of Proteomics (2018): n. pag. Web.

Jha, Kumar et al. “Concentrated Conditioned Media from Adipose Tissue Derived Mesenchymal Stem Cells Mitigates Visual Deficits and Retinal Inflammation Following Mild Traumatic Brain Injury.” International Journal of Molecular Sciences 19.7 (2018): 2016. Web.

Ungewiß, Hanna et al. “Dsg2 via Src-Mediated Transactivation Shapes EGFR Signaling towards Cell Adhesion.” Cellular and Molecular Life Sciences (2018): n. pag. Web.

Hall, Caroline L, Adrienne R Wells, and Kai P Leung. “Pirfenidone Reduces Profibrotic Responses in Human Dermal Myofibroblasts, in Vitro.” Laboratory Investigation 98.5 (2018): 640–655. Web.

Jia, Jun et al. “Fibroblast Activation Protein-α Promotes the Growth and Migration of Lung Cancer Cells via the PI3K and Sonic Hedgehog Pathways.” International Journal of Molecular Medicine (2017): n. pag. Web.

Marciel, Michael P. et al. “Selenoprotein K Deficiency Inhibits Melanoma by Reducing Calcium Flux Required for Tumor Growth and Metastasis.” Oncotarget 9.17 (2018): n. pag. Web.

Bai, Xiao Xu et al. “Downregulation of Metabotropic Glutamate Receptor 5 Inhibits Hepatoma Development in a Neurotoxin Rotenone-Induced Parkinson’s Disease Model.” Toxicology Letters 288 (2018): 71–81. Web.

Dickinson, Catherine M. et al. “Leukadherin-1 Ameliorates Endothelial Barrier Damage Mediated by Neutrophils from Critically Ill Patients.” Journal of Intensive Care 6.1 (2018): 19. Web.

Horváth, Dániel et al. “Myosin Phosphatase Accelerates Cutaneous Wound Healing by Regulating Migration and Differentiation of Epidermal Keratinocytes via Akt Signaling Pathway in Human and Murine Skin.” Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1864.10 (2018): 3268–3280. Web.

Brückner, Bastian Rouven et al. “Mechanical and Morphological Response of Confluent Epithelial Cell Layers to Reinforcement and Dissolution of the F-Actin Cytoskeleton.” Progress in Biophysics and Molecular Biology (2018): n. pag. Web.

Oberleitner, Maximilian. “QCM-ECIS: Combined Viscoelastic and Dielectric Sensing of Cells.” N.p., 2018. 151–293. Web.

Xie, Zhihui et al. “Adrenomedullin Surges Are Linked to Acute Episodes of the Systemic Capillary Leak Syndrome (Clarkson Disease).” Journal of Leukocyte Biology 103.4 (2018): 749–759. Web.

Potter, Daniel R. et al. “Mesenchymal Stem Cell-Derived Extracellular Vesicles Attenuate Pulmonary Vascular Permeability and Lung Injury Induced by Hemorrhagic Shock and Trauma.” Journal of Trauma and Acute Care Surgery 84.2 (2018): 245–256. Web.

Peinhaupt, Miriam et al. “DP1 Receptor Signaling Prevents the Onset of Intrinsic Apoptosis in Eosinophils and Functions as a Transcriptional Modulator.” Journal of Leukocyte Biology 104.1 (2018): 159–171. Web.

Alsaffar, Hiba et al. “Interleukin-6 Promotes a Sustained Loss of Endothelial Barrier Function via Janus Kinase-Mediated STAT3 Phosphorylation and de Novo Protein Synthesis.” American Journal of Physiology-Cell Physiology 314.5 (2018): C589–C602. Web.

Kiseleva, Raisa Yu. et al. “Vascular Endothelial Effects of Collaborative Binding to Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1).” Scientific Reports 8.1 (2018): 1510. Web.

Martello, Andrea et al. “Phenotypic MiRNA Screen Identifies MiR-26b to Promote the Growth and Survival of Endothelial Cells.” Molecular Therapy - Nucleic Acids 13 (2018): 29–43. Web.

Moscariello, Pierpaolo et al. “Brain Delivery of Multifunctional Dendrimer Protein Bioconjugates.” Advanced Science 5.5 (2018): 1700897. Web.

Smyth, Leon C. D. et al. “Unique and Shared Inflammatory Profiles of Human Brain Endothelia and Pericytes.” Journal of Neuroinflammation 15.1 (2018): 138. Web.

Suarez-Lopez, Lucia et al. “MK2 Contributes to Tumor Progression by Promoting M2 Macrophage Polarization and Tumor Angiogenesis.” Proceedings of the National Academy of Sciences (2018): 201722020. Web.

Rossmann, Christine et al. “Ethyl Pyruvate Inhibits Oxidation of LDL in Vitro and Attenuates OxLDL Toxicity in EA.Hy926 Cells.” Ed. Gianfranco Pintus. PLOS ONE 13.1 (2018): e0191477. Web.

Skaria, Tom, Esther Bachli, and Gabriele Schoedon. “RSPO3 Impairs Barrier Function of Human Vascular Endothelial Monolayers and Synergizes with Pro-Inflammatory IL-1.” Molecular Medicine 24.1 (2018): 45. Web.

Veeraraghavan, Rengasayee et al. “The Adhesion Function of the Sodium Channel Beta Subunit (Β1) Contributes to Cardiac Action Potential Propagation.” eLife 7 (2018): n. pag. Web.

Giana, Fabián Eduardo, Fabián José Bonetto, and Mariela Inés Bellotti. “Assay Based on Electrical Impedance Spectroscopy to Discriminate between Normal and Cancerous Mammalian Cells.” Physical Review E 97.3 (2018): 032410. Web.

McQueeney, Kelley E. et al. “Targeting Ovarian Cancer and Endothelium with an Allosteric PTP4A3 Phosphatase Inhibitor.” Oncotarget 9.9 (2018): 8223–8240. Web.

Barabutis, Nektarios et al. “Wild-Type P53 Enhances Endothelial Barrier Function by Mediating RAC1 Signalling and RhoA Inhibition.” Journal of Cellular and Molecular Medicine 22.3 (2018): 1792–1804. Web.

Leonard, Antony, Arshad Rahman, and Fabeha Fazal. “Importins α and β Signaling Mediates Endothelial Cell Inflammation and Barrier Disruption.” Cellular Signalling 44 (2018): 103–117. Web.

Serrano, Juan et al. “An Empirical-Mathematical Approach for Calibration and Fitting Cell-Electrode Electrical Models in Bioimpedance Tests.” Sensors 18.7 (2018): 2354. Web.

Kuck, Jamie L. et al. “Ascorbic Acid Attenuates Endothelial Permeability Triggered by Cell-Free Hemoglobin.” Biochemical and Biophysical Research Communications 495.1 (2018): 433–437. Web.

Andrews, Allison M et al. “Characterization of Human Fetal Brain Endothelial Cells Reveals Barrier Properties Suitable for in Vitro Modeling of the BBB with Syngenic Co-Cultures.” Journal of Cerebral Blood Flow & Metabolism 38.5 (2018): 888–903. Web.

D’Antoni, Michelle L. et al. “Cenicriviroc Inhibits Trans-Endothelial Passage of Monocytes and Is Associated with Impaired E-Selectin Expression.” Journal of Leukocyte Biology (2018): n. pag. Web.

Cooper, Denise R. et al. “Human Adipose-Derived Stem Cell Conditioned Media and Exosomes Containing MALAT1 Promote Human Dermal Fibroblast Migration and Ischemic Wound Healing.” Advances in Wound Care 7.9 (2018): 299–308. Web.

Potor, László et al. “Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction in Atherosclerotic Lesion.” Oxidative Medicine and Cellular Longevity 2018 (2018): 1–16. Web.

Bhattacharjee, Rakesh et al. “Exosomal Cargo Properties, Endothelial Function and Treatment of Obesity Hypoventilation Syndrome: A Proof of Concept Study.” Journal of Clinical Sleep Medicine 14.05 (2018): 797–807. Web.

Akla, Naoufal et al. “BMP (Bone Morphogenetic Protein) 9/Alk1 (Activin-Like Kinase Receptor Type I) Signaling Prevents Hyperglycemia-Induced Vascular Permeability.” Arteriosclerosis, Thrombosis, and Vascular Biology (2018): ATVBAHA.118.310733. Web.

Li, Liting et al. “Psoriasin Overexpression Confers Drug Resistance to Cisplatin by Activating ERK in Gastric Cancer.” International Journal of Oncology (2018): n. pag. Web.

Paez-Mayorga, Jesus et al. “Ponatinib Activates an Inflammatory Response in Endothelial Cells via ERK5 SUMOylation.” Frontiers in Cardiovascular Medicine 5 (2018): n. pag. Web.

Meegan, Jamie E. et al. “Citrullinated Histone 3 Causes Endothelial Barrier Dysfunction.” Biochemical and Biophysical Research Communications 503.3 (2018): 1498–1502. Web.

Amado-Azevedo, Joana et al. “A Functional SiRNA Screen Identifies RhoGTPase-Associated Genes Involved in Thrombin-Induced Endothelial Permeability.” PloS one 13.7 (2018): e0201231. Print.

Fallacara, Arianna et al. “Combination of Urea-Crosslinked Hyaluronic Acid and Sodium Ascorbyl Phosphate for the Treatment of Inflammatory Lung Diseases: An in Vitro Study.” European Journal of Pharmaceutical Sciences 120 (2018): 96–106. Print.

Mleynek, Tara et al. “Endothelia Extrude Apoptotic Cells to Maintain a Constant Barrier.” bioRxiv (2018): 268946. Print.

Hilfenhaus, Georg et al. “Vav3-Induced Cytoskeletal Dynamics Contribute to Heterotypic Properties of Endothelial Barriers.” J Cell Biol (2018): jcb-201706041. Print.

Pérez, Pablo et al. “Remote Cell Growth Sensing Using Self-Sustained Bio-Oscillations.” Sensors 18.8 (2018): 2550. Print.

Slater, Sadie C et al. “MicroRNA-532-5p Regulates Pericyte Function through Targeting the Transcription Regulator BACH1 and Angiopoietin-1.” Molecular Therapy (2018): n. pag. Print.

van der Vlugt, Luciën E P M et al. “Farm Dust Reduces Viral Load in Human Bronchial Epithelial Cells by Increasing Barrier Function and Antiviral Responses.” Journal of Allergy and Clinical Immunology 141.5 (2018): 1949–1952. Print.

Park, Sang-Oh et al. “Electrical Cell-Substrate Impedance Spectroscopy System Using Cloud Based Database.” 2018 IEEE International Conference on Applied System Invention (ICASI). IEEE, 2018. 267–270. Print.

Liao, Danying et al. “CRISPR-Mediated Deletion of the PECAM-1 Cytoplasmic Domain Increases Receptor Lateral Mobility and Strengthens Endothelial Cell Junctional Integrity.” Life sciences 193 (2018): 186–193. Print.

Amado-Azevedo, J et al. “The Minor Histocompatibility Antigen 1 (HMHA1)/ArhGAP45 Is a RacGAP and a Novel Regulator of Endothelial Integrity.” Vascular pharmacology 101 (2018): 38–47. Print.

Pannekoek, Willem-Jan, Marjolein J Vliem, and Johannes L Bos. “Multiple Rap1 Effectors Control Epac1-Mediated Tightening of Endothelial Junctions.” Small GTPases (2018): 1–8. Print.

Pati, Shibani et al. “Lyophilized Plasma Attenuates Vascular Permeability, Inflammation and Lung Injury in Hemorrhagic Shock.” PloS one 13.2 (2018): e0192363. Print.

Kiewiet, Mensiena B G et al. “The Epithelial Barrier-Protecting Properties of a Soy Hydrolysate.” Food & function 9.8 (2018): 4164–4172. Print.

Sun, Xu et al. “Anti‑cancer Effects of Fisetin on Mammary Carcinoma Cells via Regulation of the PI3K/Akt/MTOR Pathway: In Vitro and in Vivo Studies.” International journal of molecular medicine 42.2 (2018): 811–820. Print.

Leligdowicz, Aleksandra et al. “Human Pulmonary Endothelial Cell Permeability after Exposure to LPS-Stimulated Leukocyte Supernatants Derived from Patients with Early Sepsis.” American Journal of Physiology-Lung Cellular and Molecular Physiology (2018): n. pag. Print.

Kovačević, Igor et al. “The Cullin-3–Rbx1–KCTD10 Complex Controls Endothelial Barrier Function via K63 Ubiquitination of RhoB.” The Journal of Cell Biology 217.3 (2018): 1015–1032. Web.

Lenin, Raji et al. “Critical Role of Endoplasmic Reticulum Stress in Chronic Endothelial Activation− Induced Visual Deficits in Tie2‐tumor Necrosis Factor Mice.” Journal of cellular biochemistry (2018): n. pag. Print.

Wu, Li et al. “Vascular Endothelial Growth Factor Aggravates Cerebral Ischemia and Reperfusion-Induced Blood-Brain-Barrier Disruption through Regulating LOC102640519/HOXC13/ZO-1 Signaling.” Experimental cell research (2018): n. pag. Print.

Kovacs‐Kasa, Anita et al. “Extracellular Adenosine‐induced Rac1 Activation in Pulmonary Endothelium: Molecular Mechanisms and Barrier‐protective Role.” Journal of cellular physiology 233.8 (2018): 5736–5746. Print.

Cvitic, Silvija et al. “Human Fetoplacental Arterial and Venous Endothelial Cells Are Differentially Programmed by Gestational Diabetes Mellitus, Resulting in Cell-Specific Barrier Function Changes.” Diabetologia (2018): 1–14. Print.

Robilliard, Laverne et al. “The Importance of Multifrequency Impedance Sensing of Endothelial Barrier Formation Using ECIS Technology for the Generation of a Strong and Durable Paracellular Barrier.” Biosensors 8.3 (2018): 64. Print.

Troletti, Claudio Derada et al. “Notch Signaling Is Impaired during Inflammation in a Lunatic Fringe-Dependent Manner.” Brain, behavior, and immunity 69 (2018): 48–56. Print.

Cavallini, F., and M. Tarantola. “ECIS Based Wounding and Reorganization of Cardiomyocytes and Fibroblasts in Co-Cultures.” Progress in Biophysics and Molecular Biology (2018): n. pag. Web.

Bhargavan, Biju, and Georgette D. Kanmogne. “Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis.” Molecular Neurobiology 55.2 (2018): 1352–1363. Web.

Szulcek, Robert et al. “The Covalently Immobilized Antimicrobial Peptide LL37 Acts as a VEGF Mimic and Stimulates Endothelial Cell Proliferation.” Biochemical and biophysical research communications 496.3 (2018): 887–890. Print.

Kostadinova, Elena et al. “NLRP3 Protects Alveolar Barrier Integrity by an Inflammasome-Independent Increase of Epithelial Cell Adherence.” Scientific Reports 6.1 (2016): 30943. Web.

Zou, Qing‐yun et al. “GNA11 Differentially Mediates Fibroblast Growth Factor 2‐and Vascular Endothelial Growth Factor A‐induced Cellular Responses in Human Fetoplacental Endothelial Cells.” The Journal of physiology (2018): n. pag. Print.

Curtis, Theresa M. et al. “The Secretome of Adipose-Derived Mesenchymal Stem Cells Protects SH-SY5Y Cells from Arsenic-Induced Toxicity, Independent of a Neuron-like Differentiation Mechanism.” NeuroToxicology 67 (2018): 54–64. Web.

Ghadiri, Maliheh et al. “The Use of Fatty Acids as Absorption Enhancer for Pulmonary Drug Delivery.” International Journal of Pharmaceutics 541.1–2 (2018): 93–100. Web.

Shaver, Ciara M. et al. “Cell-Free Hemoglobin Promotes Primary Graft Dysfunction through Oxidative Lung Endothelial Injury.” JCI Insight 3.2 (2018): n. pag. Web.

Chandra, Deepak et al. “Dimethylarginine Dimethylaminohydrolase (DDAH) Overexpression Enhances Wound Repair in Airway Epithelial Cells Exposed to Agricultural Organic Dust.” Inhalation Toxicology 30.3 (2018): 133–139. Web.

Vila Cuenca, Marc et al. “Stabilization of Cell–Cell Junctions by Active Vitamin D Ameliorates Uraemia-Induced Loss of Human Endothelial Barrier Function.” Nephrology Dialysis Transplantation (2018): n. pag. Web.

Chiang, Nan et al. “New Maresin Conjugates in Tissue Regeneration Pathway Counters Leukotriene D 4 –Stimulated Vascular Responses.” The FASEB Journal 32.7 (2018): 4043–4052. Web.

Alma, L.J. et al. “Endothelial Dysfunction as a Long-Term Effect of Late Onset Hypertensive Pregnancy Disorders: High BMI Is Key.” European Journal of Obstetrics & Gynecology and Reproductive Biology 225 (2018): 62–69. Web.

Xiao, Bo et al. “TNFα Gene Silencing Mediated by Orally Targeted Nanoparticles Combined with Interleukin-22 for Synergistic Combination Therapy of Ulcerative Colitis.” Journal of Controlled Release (2018): n. pag. Web.

Ghavampour, Sharang et al. “Endothelial Barrier Function Is Differentially Regulated by CEACAM1-Mediated Signaling.” The FASEB Journal (2018): fj.201800331R. Web.

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!