Analysis of plasma membrane integrity by fluorescent detection of Tl(+) uptake

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 efficiently detected and studied by fluorescent measurement of Tl(+) uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl(+)-sensitive fluorophore FluxOR and subjected to USEPs in a Tl(+)-containing bath buffer, displayed an immediate (within <100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl(+) by 600-ns USEP was at 1-2 kV/cm, and the rate of Tl(+) uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1-1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca(2+) did not block the USEP effect. Addition of a Tl(+)-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent 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.