Fig 1.
Experimental setup for electrostatic actuation of a water droplet in dielectric oil.
Overview (A), top view (B), and side view (C) of the experimental apparatus. * in B indicates connection to a DC HV power supply. The numerical value in C indicates the gap between the electrodes, in millimeters.
Fig 2.
Behavior of Jurkat cells in a droplet during bouncing.
A 3.0 μl aliquot containing 1.0 × 105 Jurkat cells in RPMI-1640 without serum was added to the silicone oil. A charge of 1.5 kV was applied to the electrodes, with a 5.08 mm gap, to initiate droplet bouncing. High voltage was applied for 10 minutes to continue the droplet bouncing. A: Initial state of the droplet bouncing. B: After 10 minutes of droplet bouncing.
Fig 3.
Cell viability and fluorescent protein expression measured using flow cytometry.
Jurkat cells were treated with droplet bouncing (1.5 kV, 10 minutes) or short-circuiting via the droplet (2.5 or 3.0 kV, 1 short) and assayed according to 7-AAD uptake for cell death 24 hours after treatment. The electrode gap was 5.08 mm. “Control” indicates a control experiment, in which the droplet containing cells and plasmid DNA was added to the oil and recovered immediately. A: Intensity of fluorescence of YFP and 7-AAD was measured, and plotted using a log scale. The flow cytometry data are displayed as density plots. The plot is divided into four quadrants (Q1, Q2, Q3, and Q4). Quadrants Q1 and Q2 include the 7-AAD positive (dead) cells, Q3 includes YFP negative and 7-AAD negative cells, and Q4 includes YFP positive and 7-AAD negative cells. The percentages of 7-AAD positive (dead) cells (Q1+Q2) and YFP-positive viable cells (Q4) are shown. Results are representative of at least three independent experiments. B: Transfection efficiency and cell viability 24 hours after droplet bouncing or short-circuiting, as determined using flow cytometry. Data are expressed as the mean ± standard deviation (SD) of triplicate measurements. Statistical significance was determined using Student’s t-tests, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. ctrl. C: Effect of the number of short-circuits. The voltage was set to 3.0 kV. Data are expressed as the mean ± SD of triplicate measurements. Statistical significance was determined using Student’s t-tests, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. ctrl.
Fig 4.
Permeabilization of YO-PRO-1 molecules measured by flow cytometry.
A: YO-PRO-1 uptake was induced by short-circuiting via the droplets (2.5 or 3.0 kV, 1 short). The fluorescence intensities were determined for YO-PRO-1 and 7-AAD, and then plotted using a log scale. The flow cytometry data are displayed as density plots. The plot is divided into four quadrants (Q1, Q2, Q3, and Q4). Quadrants Q1 and Q2 contain 7-AAD positive (dead) cells, Q3 contains YO-PRO-1 negative and 7-AAD negative cells; Q4 contains YO-PRO-1 positive and 7-AAD negative cells. The percentages of 7-AAD positive (dead) cells (Q1+Q2) and YO-PRO-1-positive viable cells (Q4) are shown. Results are representative of at least three independent experiments. B: YO-PRO-1 uptake efficiency and cell viability one hour after short-circuiting, as determined using flow cytometry. Data are expressed as the mean ± SD of at least three measurements. Statistical significance was determined using Student’s t-tests, **p < 0.01, ***p < 0.001 vs. ctrl. C: YO-PRO-1 uptake was not stimulated by droplet bouncing for 10 minutes. The result is representative of four independent experiments. D: YO-PRO-1 uptake efficiency and cell viability one hour after droplet bouncing. Jurkat cells were treated with droplet bouncing for 10 minutes with the indicated electric field strength. Data are expressed as the mean ± SD of at quadruplicate measurements. E: Gene transfection by droplet bouncing with a more intense electric field strength. Data are expressed as the mean ± SD of triplicate measurements.
Fig 5.
Influx of calcium ions measured by flow cytometry.
A: The influx of Ca2+ was stimulated by short-circuiting via the droplets (3.0 kV, 1 or 5 shorts). A 3.0 μl droplet containing 1.0×105 Fluo-4-loaded Jurkat cells suspended in HEPES-buffered saline with (+) or without (-) 10 mM CaCl2 was added to the silicone oil, and short-circuiting via a droplet was performed. (A-1) Typical flow cytometry histograms. The fluorescence intensity was determined for Fluo-4. The flow cytometry histogram of the untreated control is filled gray. The black dashed line indicates the threshold of the Fluo-4 positive cells. The percentage of the Fluo-4 positive cells is shown. Results are representative of at least three independent experiments. (A-2) The population of Fluo-4 positive cells, as determined using flow cytometry. Data are expressed as the mean ± SD of at least three measurements. Statistical significance was determined using Student’s t-tests, *p < 0.05, **p < 0.01, ***p < 0.001 vs. ctrl. B: Droplet bouncing (6.56 kV/cm, 10 minutes) did not increase Fluo-4 fluorescence intensity. (B-1) Typical flow cytometry histograms. A 3.0 μl droplet containing 1.0×105 Fluo-4-loaded Jurkat cells suspended in HEPES-buffered saline with 10 mM CaCl2 was added to the silicone oil, and droplet boncing was performed. The flow cytometry histogram of the untreated control is filled gray. The result is representative of four independent experiments. (B-2) Median Fluo-4 fluorescence intensity, as determined using flow cytometry. Data are expressed as the mean ± SD of at least three measurements.
Fig 6.
Measurement of endocytosis induced by short-circuiting via an aqueous droplet.
A: Effect of pre-treatment of cells with endocytosis inhibitors on gene transfection. Jurkat cells were pre-treated with endocytosis inhibitors, MβCD (A-1) and Pitstop 2-100 (A-2), before short-circuiting (3.0 kV, 5.08 mm gap, 1 short) and assayed for cell death using 7-AAD uptake for 24 hours after droplet EP. Data are expressed as the mean ± SD of at three measurements. Statistical significance was determined using Student’s t-tests, *p < 0.05 and **p < 0.01 vs. vehicle control, and ##p < 0.01. B: Monitoring endocytosis with pH-sensitive fluorescent dextran conjugates. Typical flow cytometry histograms (B-1) and population of pHrodo Green positive cells (B-2) are shown. The black dashed line in B-1 indicates the threshold of the pHrodo Green positive cells. Data are expressed as the mean ± SD of at quadruplicate measurements. Statistical significance was determined using Student’s t-tests, ***p < 0.001 vs. untreated control.
Fig 7.
The possible mechanism for gene transfection of mammalian cells by short-circuiting via an aqueous droplet in dielectric oil.
Short-circuiting stimulates transient membrane pore formation, shown by the influx of calcium ions and YO-PRO-1 uptake. Endocytosis, especially clathrin-mediated endocytosis, is essential in exogenous gene expression. Droplet bouncing does not deliver extracellular molecules.