Freezing point microosmometer

A cover slip with cells was transferred into a glass-bottomed chamber (Warner Instruments, Hamden, CT) mounted on an Olympus IX81 inverted microscope equipped with an FV 1000 confocal laser scanning system (Olympus America, Center Valley, PA). The chamber was filled with a buffer composed of (in mM): 136 NaCl, 5 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4), with the addition of 5 µg/ml (7.5 µM) of Pr iodide and 1 µM of YP iodide. This ratio of concentrations was established empirically, in order to account for the different brightness of the dyes, enable their reliable detection, and minimize the chance of quenching (see section 3.2). The same concentrations are commonly used in membrane permeabilization studies with these dyes. For one experiment illustrated in Fig. 2, the buffer contained 43 µg/ml of Pr and no YP.
The buffer osmolality was at 290–300 mOsm/kg, as measured with a freezing point microosmometer (Advanced Instruments, Inc., Norwood, MA). The chemicals were obtained from Sigma–Aldrich (St. Louis, MO) and Invitrogen (Eugene, OR).
Images were acquired with a 40×, NA 0.95 dry objective. YP and Pr were excited at 488 and 543 nm, and the emission was collected at 505–525 nm and 560–660 nm, respectively. The lasers were operated in a line sequence mode to avoid the “cross-talking” of the dyes.
The sensitivity of emission detectors (photomultiplier tubes, PMT) was chosen individually for different sets of experiments. For those summarized in Fig. 1, the sensitivity was relatively low, to cover a broad range of Pr uptake with possible pixel saturation only in digitonin-permeabilized cells. For Fig. 2, the PMT sensitivity was tuned as indicated in the figure legend. For experiments in Figs. 3-6 and Fig. 8, the sensitivity was set constant at the highest level which still prevented pixel saturation after the most intense nsEP treatment (100 pulses). Experiments presented in Fig. 7 were performed separately from the rest of the study and utilized somewhat different laser and PMT settings; calibrations shown if Fig. 3 do not apply to these data.
Stacks of images captured before and after pulsing (in most experiments, at regular 10-s intervals) were quantified with MetaMorph Advanced v.7.7.10.0 (Molecular Devices, Foster City, CA).

A coverslip with cells was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) mounted on an Olympus IX71 inverted microscope equipped with an FV 1000 confocal laser scanning system (Olympus America, Center Valley, PA). The chamber was filled with a physiological solution containing (in mM): 140 NaCl, 5 KCl, 2 MgCl₂, 2 CaCl₂, 1 HEPES, 10 Glucose, X GdCl₃, (pH 7.4 with NaOH) where X was varied from 0 to 1 mM. The concentration of HEPES was kept at a minimum to prevent precipitation of Gd³⁺ in the solution. The osmolality of the solution was between 290 and 310 mOsm/kg, as measured by a freezing point microosmometer (Advanced Instruments, Inc., Norwood, MA). The membrane-impermeable fluorescent dyes YP and Pr iodide were added to the solution at 1 μM and 5μg/mL, respectively. These dyes are non-fluorescent when in the chamber solution, but once they enter the cell, their emission increases profoundly upon binding to intracellular nucleic acids (20 (link)). All chemicals were purchased from Sigma-Aldrich and Life Technologies (Grand Island, NY). Where indicated in the text, the chamber was continuously perfused with the physiological solution (including the dyes) during the experiment, with a flow rate of 3 mL/min. All experiments were performed at room temperature (22 ± 2°C).
Differential-interference contrast (DIC) and fluorescent images were taken with a 40X, NA 0.95 dry objective as a time series beginning before nsEP exposure and continuing for up to 7 minutes after it. YP was excited with a blue laser (488 nm) and Pr was excited with a green laser (543 nm); the emission of each dye was detected between 505 and 525 nm or between 560 and 660 nm, respectively. To avoid “cross-talk” between the dyes, the lasers were operated in a line sequence mode. Images were quantified using MetaMorph Advanced v.7.7.0.0 (Molecular Devices, Foster City, CA).