Mechanism of Electroporative Dye Uptake by Mouse B Cells E. Neumann, K. Toensing, S. Kakorin, P. Budde, J. Frey  Biophysical Journal  Volume 74, Issue 1, Pages 98-108 (January 1998) DOI: 10.1016/S0006-3495(98)77771-9 Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 1 Optical image of the primary data. B-lymphoma cells (line IIA1.6) exposed to one electric field pulse (E=1.23kV cm−1; pulse duration tE=110μs) in the presence of the dye SERVA blue G (Mr 854). (A) Nonpermeabilized cells (white) and permeabilized cells (blue), indicated by the uptake of the dye (phase-contrast); bar=50μm. (B) Blebs are observed (dark-contrast method) almost immediately after the application of one electric pulse of low field strength (E<Ec). Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 2 Electroporative cell coloring by the dye SERVA blue G. The fraction N0/NT of colored cells as a function of the external field strength E at two pulse durations tE/μs: 50 (□) and 150 (■). N0 is the number of blue-colored cells corresponding to the administration of SERVA blue G in the cell suspension before the pulsing, and NT is the total number of cells. The fraction Nd/NT at E=0 (dashedline) represents the number of damaged cells colored before the field application. For a comparison, the range where the cells can be transfected (shadedarea) at larger pulse durations (tE ≈ 100ms) is restricted to a narrow lower field strength range (0.6–0.7kV cm−1). Experimentally, an aliquot of a suspension of mouse B-cells (line IIA1.6) was treated by one rectangular electric pulse in the presence of 1.17mM SERVA blue G (Mr 854); the largest molecular dimension was estimated to be 2.4±1nm. CLICK's medium was based on RPMI 1640 supplemented with 10mM Na-pyruvate, 10mM glutamine, and 50μM 2-mercaptoethanol. The electric conductivity of the cell suspension is λ0=1.3×10−2 S cm−1, T=293K (20°C); cell density ρC=NT/Vo ≈ 6×106 ml−1; sample volume is Vo=800μl. Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 3 Cell resealing kinetics indicated by dye uptake. The fraction fN=(N−N∞)/(N0−N∞) of colored cells (Eq. 2) as a function of the time tadd of dye addition after the pulse of the duration tE=110μs and the external field strength E=1.49kV cm−1. (Inset) Semilogarithmic scale, showing the biphasic nature of the cell coloring. The initial value fN=1 for N=N0 (at time tadd=0) corresponds to the addition of the dye before the pulsing. Obviously, at tadd → ∞, where N=N∞, fN=0. Experimental conditions were as in Fig. 2. Data were evaluated with Eq. 23 of the text. Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 4 Optimal range for cell survival. The fraction N/NT of colored cells is expressed as a function of field strength E (pulse duration: tE=110μs). ■, N0/NT pulsed in the presence of dye. ▴, N∞/NT pulsed in the absence of dye; the dye was added 30min after the electric pulse. Dotted line, ΔN/NT=(N0−N∞)/NT; the shaded area represents the range of the experimental conditions of an optimal transient cell electroporation leading to transport of dyes like SERVA blue G. Experimental conditions were as in Fig. 2. Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 5 Strength-duration relationship. Square of the field strength (E0.52), corresponding to the fraction fN=0.5 of colored cells (see Fig. 2), is expressed as a function of the reciprocal pulse duration (1/tE) at two selected conductivities of the external medium λ0/(10−2 S cm−1): 1.3 (■) and 1.0 (□). Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 6 Kinetics of membrane electroporation. Fraction f=(N−N∞)/(NT−N∞) of those colored cells that can potentially regenerate (Eq. 1) as a function of the pulse duration tE at various external field strengths E/kV cm−1: ○, 0.64; •, 0.85; □, 1.06; ■, 1.28; △, 1.49; ▴, 1.7; ▿, 1.91; ▾, 2.13; respectively. Solid lines reflect the theoretical analysis of the data with Eq. 24. Other experimental conditions were as in Fig. 2. Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions

Figure 7 Logarithm of Kp=[P]/[C] of the overall state transition C⇌P as a function of the square of the field strength. The solid straight line indicates the theoretically predicted linear increase in ln (Kp(E2)) at a constant transmembrane conductivity. The saturation is caused by the increase in number and size of conductive pores. The increased transmembrane conductivity prevents further charging of the cell membrane. Biophysical Journal 1998 74, 98-108DOI: (10.1016/S0006-3495(98)77771-9) Copyright © 1998 The Biophysical Society Terms and Conditions