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.
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 G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 microseconds) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees 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 degrees C for 30 min ("resealing") significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 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, tau = 5 microseconds, three pulses, 4 degrees 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.
Djuzenova
C S
CS
Lehrstuhl für Biotechnologie, Universität Würzburg, Germany.
Sukhorukov
V L
VL
Klöck
G
G
Arnold
W M
WM
Zimmermann
U
U
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000528:radiation effects*; D002453:Cell Cycle / Q000528:radiation effects; D002463:Cell Membrane Permeability / Q000528:radiation effects; D048429:Cell Size; D002470:Cell Survival; D004247:DNA / Q000032:analysis; D004558:Electric Stimulation; D005434:Flow Cytometry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D011947:Receptors, Antigen, B-Cell / Q000032:analysis*
D008070:Lipopolysaccharides; D011947:Receptors, Antigen, B-Cell; D004247:DNA