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

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 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.

Fig. 1

Relative dimensions of propidium, YO-PRO-1 and thallium cations. Molecular views in two perpendicular planes were generated using Jmol open-source Java viewer for chemical structures in 3D (http://www.jmol.org/). Gray areas show van der Waals surface of impenetrable molecular volume (Creighton 1993). The scale is defined by vertical and horizontal rulers

Fig. 2

Comparative dimensions and shapes of three cell types used in Tl⁺ uptake studies. Images were taken using DIC optics. Scale bar = 20 μm for all images

Fig. 3

Concurrent detection of Tl⁺ and propidium uptake triggered by USEP exposures. In both a and b, three rows (from top to bottom) are Tl⁺-sensitive FluxOR dye emission, propidium emission and DIC images of CHO cells. Images were taken every 2 s. a Selected image triplets at indicated times from the start of the experiment. b Left panels Cell images at the onset of the experiment. Line drawn over the images denotes the position of an imaginable “slice” through the time lapse stack of 2D images. Right panels This “slice” as a time change display; i.e., they show how the image at the position of the line changed over time. The parameters of two USEP exposures are shown at the top; 1p and 10p correspond to one pulse and a train of 10 pulses, respectively. b Onset of exposures is shown by vertical dashed lines over the time change display. Note fast and profound uptake of Tl⁺ after a single pulse but only a weak and delayed uptake of propidium after 10 pulses. Bath buffers were 7 (a) and 2 (b), see Table 1. Scale bars = 10 μm

Fig. 4

Effect of sequential USEP exposures at increasing intensity on Tl⁺ uptake in NG108 cells. The intensity of FluxOR dye emission was measured every 2 s in three neuroblastoma cells in buffer 2 (Table 1). Vertical dashed lines show times of single-pulse exposures at indicated intensities. Inset Overlapped fluorescent and DIC images of one of the cells and a respective time change display of fluorescence (see Fig. 3b for detail). Scale bar = 10 μm. Note stepwise emission increases in response to USEP exposures, with the threshold between 1.5 and 2.6 kV/cm

Fig. 5

Concurrent detection of YO-PRO-1 and propidium uptake triggered by USEP exposures in CHO cells. Images were prepared the same way as in Fig. 3b, but the top row is YO-PRO-1 emission. a Only 12-kV/cm pulses triggered dye uptake. b Additional cell images at right show that uptake of both tested dyes started from the right-hand top side of the cell (this was the side facing the positive USEP-delivering electrode, not shown). Bath buffers were 1 (a) and 2 (b), see Table 1. Scale bars = 20 μm (a) and 10 μm (b)

Fig. 6

Membrane permeabilization by USEPs at different electric field intensities as quantified by Tl⁺ uptake. a CHO cells were bathed in Tl-CB (buffer 2, Table 1), and fluorescent images were taken every 2 s. A single 600-ns pulse was applied at 40 s; the electric field intensity is indicated to the right of the graphs (20–35 cells from 5–10 independent experiments per each condition). For clarity, error bars are shown in one direction only. The initial (pre-exposure) emission value was taken as zero, and the mean steady-state emission value after the most intense exposure (10.2 kV/cm) was taken as 100%. Note that the initial rise in fluorescence intensity immediately after USEP was practically linear; for clarity, the best-fit linear function is shown for 10.2 kV/cm exposure only (dashed line). The slope of this line reflects the initial rate of Tl⁺ uptake and was used in b as a measure of membrane permeabilization. The original data for GH3 cells are not shown but are similar to those for CHO cells in a. Note that the rate of Tl⁺ uptake increased almost linearly with increasing electric field, with the threshold for both cell lines between 1 and 2 kV/cm

Fig. 7

Effect of bath buffer composition on USEP-triggered Tl⁺ uptake. CHO cells were exposed at 40 s to a single 600-ns pulse at 0 (control), 3.9 or 6.6 kV/cm in different buffers (20–30 cells per condition). Bath buffers, from left to right panels, were 2, 3, 4 and 5, respectively (see Table 1). Principal changes to buffer composition are outlined in boxes next to graphs. In short, buffer 2 (left panel) contained Cs⁺ and Ca²⁺ ions and K⁺-channel blockers TEA and 4-AP. For experiments in the second panel, Ca²⁺ was removed from the buffer. Third panel TEA and 4-AP were also removed. Right panel Cs⁺ was also removed and replaced with Na⁺

Fig. 8

Timeline of nanopore resealing as visualized by gradual reduction of Tl⁺ uptake with time after USEP exposure. NG108 cells were placed in a Tl⁺ -free buffer (6, Table 1) and subjected to a single 600-ns pulse at either 6.6 kV/cm (top graphs) or 0 kV/cm (“sham” exposure, bottom graphs); 8–11 cells per group. At indicated time intervals after USEP, the bath perfusion was switched to buffer 5, containing 16 mM of Tl⁺. Uptake of Tl⁺ caused a surge of cell fluorescence, which was more profound if nanopores were still open. See text for more detail

Fig. 9

Fast onset of USEP-triggered Tl⁺ uptake. Shown are individual data for three CHO cells (inset) bathed in Tl-CB (buffer 2, Table 1). Images were taken every 130 ms, with a scanning time of about 40 ms per image. Note diffuse Tl⁺ entry (inset), in contrast to single pole entry of other dyes (Fig. 5b). See text for more detail