Journal: Cellular and molecular life sciences : CMLS

Article Title: Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

doi: 10.1007/s00018-016-2434-4

Figure Lengend Snippet: Streamlined pulsed electric field (PEF) treatment of adherent cells on coverslips with an electroconductive indium tin oxide (ITO) layer. a Standard sequence of procedures for PEF exposure in electroporation cuvettes for suspension-grown cells (left) and adherent cells (center). The use of ITO coverslips (right) eliminates multiple steps which may affect cell survival. b Schematic explaining PEF delivery to cells in an electroporation cuvette (i) with buffer (b) placed in a gap between two electrodes (e). Panels ii and iii show the enlarged view of this gap (not to scale) and the electric field lines when cells are attached to a “regular” glass coverslip (ii) or attached to an ITO-coated coverslip (iii). The regular glass coverslip shields cells from the electric field, whereas the ITO layer serves as an electrode and cancels shielding; see text for more details. c Exposure setup used in this study. The magnified area shows the electroporation cuvette tilted to keep the ITO coverslip resting flat on the bottom electrode. The ITO surface with cells is facing up into the medium. d Shape of a 300-ns pulse at 600 V (1.8 kV/cm)

Article Snippet: We utilized 8 mm diameter, # 1.5 thickness glass coverslips covered with ITO to the sheet resistance of 8–12 Ohms/sq by Diamond Coatings Ltd. (Halesowen, UK).

Techniques: Sequencing, Electroporation, Suspension

Journal: Cellular and molecular life sciences : CMLS

Article Title: Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

doi: 10.1007/s00018-016-2434-4

Figure Lengend Snippet: Analysis of survival curves after 300-ns PEF exposure. a Survival curves shown in Fig. 2 share a similar shape which is indicative of three distinct regions: the initial resistivity shoulder, logarithmic survival decline, and the residual resistivity. For quantitation of PEF sensitivity, the logarithmic decline region can be fit with a log function. b Representative log fits (dashed lines) using the survival data for IMR-32, Hep G2, and HPAF-II cells. Shaded areas define the 95% confidence intervals for each fit. LD50 was determined as the best fit value at 50% survival; the confidence interval for LD50 is the width of the shaded area at 50% survival level. When the lower and upper limits of the confidence interval were at a different distance from the mean, the larger of the two values was used. c LD50 for tested cell lines, measured as outlined in a and b. The error bars are the confidence intervals; the lack of error bar overlap indicates the significance of differences at p < 0.05 or better, see Table 1 for numerical data. d Imaging confirms the presence of survivors even after the maximum tested PEF exposure (2000 pulses, 300 ns, 1.8 kV/cm, 50 Hz). Representative DIC (top) and fluorescence images (bottom) from the center of ITO coverslips at 22–24 h after PEF exposure in indicated cell lines. Dead cells were stained with PI (red), and live cells were stained with Hoechst (blue). Insets show magnifications of selected areas

Article Snippet: We utilized 8 mm diameter, # 1.5 thickness glass coverslips covered with ITO to the sheet resistance of 8–12 Ohms/sq by Diamond Coatings Ltd. (Halesowen, UK).

Techniques: Quantitation Assay, Imaging, Fluorescence, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types

doi: 10.1007/s00018-016-2434-4

Figure Lengend Snippet: nsPEF sensitivity has no apparent correlation with cell shape, size, or growth pattern (a) or with nucleus size (b). a Representative images of monolayers of IMR-32, MRC-5, HT-1080, and HPAF-II cells on ITO coverslips in control samples (no PEF exposure). Cell nuclei were labeled with Hoechst dye (blue), and fluorescence was laid over DIC images. Cells most sensitive to nsPEF are at the top and the least sensitive at the bottom. b Bar plots showing the frequency distributions of the nuclear diameters in the same cell lines; 2000–3000 cells were measured per group. MRC-5 cells, which have an intermediate sensitivity to nsPEF, had the largest diameter nuclei

Article Snippet: We utilized 8 mm diameter, # 1.5 thickness glass coverslips covered with ITO to the sheet resistance of 8–12 Ohms/sq by Diamond Coatings Ltd. (Halesowen, UK).

Techniques: Control, Labeling, Fluorescence