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J Membrane Biol (2010) 236:1526 DOI 10.1007/s00232-010-9269-y
Analysis of Plasma Membrane Integrity by Fluorescent Detection of Tl+ Uptake
Angela M. Bowman Olena M. Nesin
Olga N. Pakhomova Andrei G. Pakhomov
Received: 18 December 2009 / Accepted: 11 June 2010 / Published online: 11 July 2010 Springer Science+Business Media, LLC 2010
Abstract The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efciently detected and studied by uorescent measurement of Tl? uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl?-sensitive uorophore FluxORTM and subjected to USEPs in a Tl?-containing bath buffer, displayed an immediate (within \100 ms), dose-dependent surge of uorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl? by 600-ns USEP was at 12 kV/cm, and the rate of Tl? uptake increased linearly with increasing the electric eld. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 11.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca2? did not block the USEP effect. Addition of a Tl?-containing buffer within less than 10 min after USEP also caused a uorescence surge, which conrms the minutes-long lifetime of nanopores. Overall, the technique of uorescent detection of Tl? uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.
Keywords Electroporation Nanosecond electric pulses
Nanopores Thallium Cell membrane Dye uptake
Membrane integrity
Introduction
In all living cells, the integrity of the plasma membrane is fundamentally important for maintenance of the specic intracellular milieu and support of various cell functions. Disruption of the membrane barrier function, as conveniently visualized by cell uptake of certain dye molecules, is routinely used as a simple and reliable criterion to distinguish between live and dead cells. Some of the most popular membrane integrity marker dyes, like propidium iodide (PI) and ethidium homodimer, increase the emission intensity by more...