Figure 1.
Simulated chemical concentration and electric field distribution of the dual-field chip.
(A) The geometry and EGF concentration distribution in the dual field chip. Note that even for a small protein like EGF, only a small portion of it would be transported and create localized chemical gradient. Regions for cell observation are outlined in yellow dash lines. (B) The dcEF strength distribution in the dual field chip. Note that cell electrotaxis and random migration (control) with and without EF can be studied in a single chip. Regions with uniform EF distribution are outlined in red dash lines.
Figure 2.
Setup of the electrotaxis experiment using XLEFC.
(A) XLEFC is composed of the top assembly, the frame of the cell culture chamber, and a TCPS dish. Stable temperature is provided by water bath. Electric field stimulation is applied by a DC power supply through Ag/AgCl plate electrodes. (B) A photo picture showing the setup on a general laboratory bench.
Figure 3.
Detailed configuration of XLEFC.
(A) The top view of the XLEFC top assembly design. The electrical current enters from the salt bridge connections through the current rectifying chamber and the gating slit, and then into the culture chamber. (B) The side view of XLEFC showing the integration of the top assembly, the cell culture chamber, and the TCPS dish. The flow direction of chloride ions, which are driven by the external dcEF, is shown in grey arrows. (C) The frame of the cell culture chamber has five layers, including one layer of soft-acrylic backbone, two layers of fluoroplastic tape, and two layers of double-sided tape (dstape). The design enables airtight seal during the experiment and quick release after the experiment.
Figure 4.
The migration trajectories of CL 1-5 cells for 2 hours in serum-containing medium.
(A) CL 1-5 under 300mV/mm dcEF shows anodal electrotaxis. Cell tracks with end positions to the right appear in red and those to the left appear in black. (B) The random migration of CL 1-5 cells without dcEF stimulation.
Figure 5.
Electrotaxis of CL 1-5 cells under various conditions.
(A) The directedness of CL 1-5 cell electrotaxis and random migration in medium with or without 4nM Erbitux and with or without EGF stimulations. (B) The speed of CL 1-5 cell electrotaxis and random migration in medium with or without 4nM Erbitux and with or without subsequent EGF stimulation.
Figure 6.
The homogeneous electric field distribution in XLEFC.
(A) The 3D model of electrolyte-filled XLEFC built using COMSOL. (B) The vector volume plot shows the rectifying effect of electric current in the rectifying chamber. Uniform electric current is obtained at the gating slits. (C) The simulated EFS distribution at the bottom of the cell culture chamber shows extremely uniform dcEF in the cell culture chamber. (D) The plot of measured EFS taken in the cell culture chamber, excluding the gating slits.
Figure 7.
The homogeneous temperature distribution in XLEFC.
(A) The infra-red thermo-image of XLEFC before dcEF stimulation. (B) The infra-red thermo-image of XLEFC after 2 hours of dcEF stimulation. (C) The plot of temperature measurement in XLEFC before dcEF stimulation. (D) The plot of temperature measurement in XLEFC after 2 hours of dcEF stimulation.
Figure 8.
(A) The phase-contrast microphotograph of CL 1-5 cells before dcEF stimulation and (B) after 2 hours of dcEF stimulation. Typical perpendicular electro-alignment was observed. Scale bar: 100µm.
Figure 9.
The RTK activation and phosphorylation of CL1 cells determined by the PathScan RTK kit.
(A) Bar chart showing the level of RTK activation in CL1 cells with and without dcEF stimulation. No tyrosine phosphorylation of the 28 RTKs was detected in CL1 cells under dcEF stimulation. (B) Bar chart showing the level of phosphorylation of four intracellular signaling nodes in CL1 cells with and without dcEF stimulation. CL1-0 cells show time-dependent phosphorylation decrease in rpS6 while CL 1-5 cells show phosphorylation increase in rpS6 and Akt axis.
Figure 10.
The Western blotting result of CL1 cells under different conditions.
(A) Under different period of dcEF stimulation. Note that CL 1-5 cells have high EGFR expression in comparison to CL1-0 cells but neither cells showed EGFR phosphorylation at Tyr1068 under dcEF stimulation. (B) CL1-0 cells and CL 1-5 cells stimulated with 20 ng/mL EGF and CL 1-5 cells stimulated under dcEF in serum-containing medium. Note that even in serum-containing medium, CL 1-5 cells show no EGFR Tyr1068 phosphorylation under dcEF stimulation, contrary to EGF-stimulated cells. The numbers below each protein band indicate the relative densitometry intensity of the protein in different conditions compared to that in the CL 1-5 cells in the control condition.