8w1e electric cell–substrate impedance sensing (ecis) cultureware™ disposable electrode arrays  (Applied BioPhysics)

Journal: Molecular Vision

Article Title: Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function

doi:

Figure Lengend Snippet: The impact of ZEB1 reduction on cell migration in HCEnC-21T cells. A : HCEnC-21T cells were transfected with either ZEB1 siRNA-C or scrambled siRNA (sc-siRNA), and knockdown of ZEB1 was validated with western blotting. B : Transfected HCEnC-21T cells were seeded to 100% confluence on an electric cell–substrate impedance sensing (ECIS) electrode array. A high current (arrow) was applied to the circular electrode underneath the central region of the cell monolayer, thus clearing the circular area. Migration of cells into the cleared area was monitored for 12 h after wounding by measuring impedance (ohms) across the electrode at 4,000 Hz. Three wells for each condition were measured; n = 3. Error bars = standard error of the mean (SEM).

Article Snippet: 8W1E electric cell–substrate impedance sensing (ECIS) Cultureware™ disposable electrode arrays (Applied BioPhysics, Troy, NY) were stabilized with F99 medium according to the manufacturer’s protocol and then were coated with 40 μg/cm 2 chondroitin sulfate A (Sigma-Aldrich) and 400 ng/cm 2 laminin (Sigma-Aldrich) in PBS for 2 h. Twenty-four hours after transfection, the HCEnC-21T cells were reseeded at 100% confluency in the stabilized electrode arrays.

Techniques: Migration, Transfection, Western Blot, Electric Cell-substrate Impedance Sensing

Journal: Molecular Vision

Article Title: Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function

doi:

Figure Lengend Snippet: The impact of ZEB1 reduction on cell barrier function in in HCEnC-21T cells. A : HCEnC-21T cells were transfected with ZEB1 siRNA-C or scrambled siRNA (sc-siRNA), and knockdown of ZEB1 was confirmed with western blotting. B : Impedance (ohm) at 4,000 Hz, interendothelial resistance (Rb), cell attachment resistance (alpha), and plasma membrane capacitance (Cm) were measured for 48 h after seeding on electric cell–substrate impedance sensing (ECIS) electrode arrays. Three wells for each condition were measured; n = 3. Error bars = standard error of the mean (SEM). Statistical significance (p<0.05) is noted for a range when two or more sequential time points demonstrated statistical significance.

Article Snippet: 8W1E electric cell–substrate impedance sensing (ECIS) Cultureware™ disposable electrode arrays (Applied BioPhysics, Troy, NY) were stabilized with F99 medium according to the manufacturer’s protocol and then were coated with 40 μg/cm 2 chondroitin sulfate A (Sigma-Aldrich) and 400 ng/cm 2 laminin (Sigma-Aldrich) in PBS for 2 h. Twenty-four hours after transfection, the HCEnC-21T cells were reseeded at 100% confluency in the stabilized electrode arrays.

Techniques: Transfection, Western Blot, Cell Attachment Assay, Electric Cell-substrate Impedance Sensing

Journal: The Journal of Cell Biology

Article Title: VEGF and Angiopoietin-1 exert opposing effects on cell junctions by regulating the Rho GEF Syx

doi: 10.1083/jcb.201207009

Figure Lengend Snippet: Ang1 and VEGF have opposing effects on Syx localization. (A) Effects of Angiopoietin-1 (Ang1) treatment (50 ng/ml for 45 min) on endogenous Syx, VE-cadherin (VE-cad), and ZO1 localization in nontarget ( control [ Ctrl ] shRNA) versus Syx-depleted ( Syx shRNA1) HUVECs. The nuclear ZO1 staining is an artifact of the rabbit antibody used here to visualize ZO1. (B) Quantification of the mean intensity of ZO1 at the junctions using ImageJ software (means ± SEM [error bars]; ∼30 cells per field; n = 6; **/ ++ , P < 0.001, compared with control kinase dead + PBS). (C) Effects of Ang1 treatment on the impedance of Syx-depleted HUVEC monolayers. Nontarget ( control shRNA) versus Syx-depleted ( Syx shRNA1) HUVECs were grown in ECIS Cultureware Electrode Arrays for 48 h (as in ). Control (PBS) and Ang1 treatments were administered simultaneously as indicated, and impedance was measured every 180 s for the duration of the experiment. Results are representative of three independent experiments performed in triplicate. (means ± SEM). (D) Effects of Ang1 alone (45 min), VEGF alone (30 min), or cotreatment with Ang1 and VEGF (15 min with Ang1 followed by 30 min with VEGF) on Syx, ZO1, and VE-cadherin localization in HUVECs. (E) Quantification of the mean intensity of Syx at the junctions using ImageJ software and expressed as a percentage of control (PBS; means ± SEM; ∼10 cells per field; n = 6; **/ ++ , P < 0.001, compared with PBS). (F) Effects of Ang1 alone (50 ng/ml), VEGF alone (50 ng/ml), or cotreatment with Ang1 and VEGF (15-min Ang1 pretreatment followed by VEGF, see arrows) on the impedance of quiescent HUVEC monolayers. Impedance values were normalized to the initial values at the beginning of the experiment and are plotted as relative impedance where the baseline value is set at 1.0 (means ± SEM). Results are representative of three independent experiments performed in triplicate. (G) Leakage of Evans blue dye from the skin of syx +/+ and syx −/− mice in response to injections of PBS, 10 ng/ml VEGF-A 165 , or 50 ng/ml Ang1 as seen in images of the injections sites. The mean leakage in response to each type of injection is shown in the histogram (means ± SEM; n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 20 µm.

Article Snippet: In brief, cells (HUVECs, 10 5 cells/well; primary mouse ECs, 5 × 10 4 cells/well) were plated in ECIS Cultureware Disposable Electrode Arrays (8W10E; Applied BioPhysics) precoated with collagen.

Techniques: Control, shRNA, Staining, Software, Injection