Effect of electric field pulses on the viability and on the membrane‐bound immunoglobulins of LPS‐activated murine B‐lymphocytes: Correlation with the cell cycle

Abstract

The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of GO/G1 cells.After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant T = 5-40 ps) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4°C or 20°C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37°C for 30 min ("resealing") significantly decreased the percentage of permeabilized cells. Electropulsed GO/Gl cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing.The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, 'c = 5 ps, three pulses, 4°C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. Key terms: Cell cycle, deconvolution, electric field effects, flow cytometry, FITC, membrane breakdown, propidium iodide, resealing, trypan blueThe treatment of cells with electric pulses is now widely used in biotechnology, medicine, and basic research for the electroinjection of foreign molecules, such as drugs, proteins, plasmids, etc., and also for electrofusion (2,13,19,20,27,37-39,41). Important applications are (a) the use of electrically loaded cells for target-specific drug administration, (b) somatic hybridization of plant protoplasts, and (c) the electrically mediated production of human hybridomas by immortalisation of a small numbers of lymphocytes (12,21,29,36).The number and viability of the hybrids and of the electrically loaded cells depend critically-among other parameters-on the peak field strength E, of the applied field pulse, and on the cell radius a. For pulses which are much longer than the charging time of the membrane, about 0.1 ps for lymphocytes in the "pulse medium" (conductivity 3.3 mS/cm), the induced membrane voltage is given by (18) 'Research funded by grants of the Deutsche Forschungsgemeinschaft (SFB 176B5 to U.Z. and W.M.A.).

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

“…The 570 nm absorbance of the samples (including light scattering) was maintained at about 0.1. The time course of red fluorescence was followed continuously (resolution 0.1 s) from 1 min before to [10][11][12][13][14][15] min after field application (recording mode of the spectrophotometer).…”

“…Simultaneous measurement of PI and CF fluorescence emitted by control or electromanipulated cells was performed in a Fluvo-Metricell flow cytometer (HEKA elektronik, Lambrecht, Federal Republic of Germany; for details, see 14). The red channel of the cytometer was calibrated for intracellular PI content.…”

“…c o s @ . ( 1 ) This is supported by the observation that, in size-heterogeneous cell populations (e.g., lipopolysaccharide-stimulated mouse B-lymphocytes), large cells in G2/M phase of the cell cycle are selectively destroyed (14).…”

“…Membrane breakdown takes place when the generated membrane potential ( Vg) superimposed on the resting transmembrane potential difference reaches or exceeds about 1 V at room temperature (7). In the case of a spherical cell, Vg depends on the field strength (E,), cell radius (a), and the angle (0) between vector E, and the line joining the cell center to the given membrane site (20):This is supported by the observation that, in size-heterogeneous cell populations (e.g., lipopolysaccharide-stimulated mouse B-lymphocytes), large cells in G2/M phase of the cell cycle are selectively destroyed (14).However, quantitative consideration of Equation 1 shows that the variability in electrosensitivity of the cell population cannot arise only from cell size heterogeneity. Because the permeabilized cell surface [ (34)] increases approximately with the square of radius, the variability in A, should be nearly twice that in the radius.…”

Role of peroxide in AC electrical field exposure effects on Friend murine erythroleukemia cells during dielectrophoretic manipulations

“…Clearly, it is important to determine whether such field exposure induces undesirable effects on the cells, and if so, to identify conditions that avoid or minimize these negative effects. Extensive studies have been reported for cell field exposure to pulse and DC electrical fields applicable to electroporation and cell fusion [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. These studies have shown that field exposure can alter cell membrane potential and membrane structure [28,[31][32][33], cause cells to deform [28,30,[32][33][34], increase cell membrane permeability [32][33][34]39], lead to reversible and irreversible dielectric breakdown of the cell membrane [26,27,39], and cause cellular DNA damage [36].…”