Abstract
Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.
Highlights
Electroporation, or pulsed electric field (PEF) treatment, is currently widely used for many applications in several cell types, including bacteria, microalgae, mammalian cells and plant cells or tissue[1,2]
Membrane permeability of L. plantarum WCFS1 was evaluated for various electric field strengths by evaluating propidium iodide (PI) uptake before and after Pulsed electric field (PEF) treatment
L. plantarum WCFS1 became permeable for PI after PEF treatment using an electric field strength of 7.5 kV/cm or higher when 2 pulses of 100 μs were applied (Fig. 2A)
Summary
Electroporation, or pulsed electric field (PEF) treatment, is currently widely used for many applications in several cell types, including bacteria, microalgae, mammalian cells and plant cells or tissue[1,2]. PI is a small (668 Da) hydrophilic fluorescent probe that can enter the cell only when the membrane integrity is compromised, for example because of pore formation during PEF treatment[11] When it binds to nucleic acids www.nature.com/scientificreports inside the cell, the red fluorescence of PI increases 20 to 30 fold, making it a valuable tool to detect membrane permeability and in some cases cell viability[11]. Many studies used this method to detect irreversible pore formation in bacteria by addition of the fluorescent stain after the PEF treatment[12,13,14,15]. These two staining methods are compared for different PEF treatments, and in addition the effect of the different PEF treatments on membrane permeability is discussed
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