Supporting Information
DIFFERENTIAL DIELECTRIC RESPONSES OF CHONDROCYTE AND JURKAT CELLS IN ELECTROMANIPULATION
BUFFERS
Ahmet C. Sabuncu1,*, Anthony J. Asmar2, 3, Michael W. Stacey2, and Ali Beskok1
1
Department of Mechanical Engineering, Southern Methodist University, Dallas, VA, 75275, USA
2
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, 23529, USA.
3
Department of Biological Sciences, Old Dominion University, Norfolk, VA, USA.
* Corresponding author
Keywords: Clausius-Mossotti Factor, Dielectrophoretic Separation, Dielectric Spectroscopy, Microfluidics,
Membrane Biophysics.
Corresponding author: Tel.: +1 214 768 3200. Fax: +1 214 768 1473. E-mail address: [email protected].
S1 Supplementary Materials and Methods
S1.1 Device Fabrication
The electrode geometry is defined by standard photolithography techniques. Gold electrodes were generated by
sputtering a thin seeding layer of chromium and a layer of gold (~ 20 nm) on standard glass slides, followed by a
lift-off process using acetone. PDMS, with a height of 500 µm, and glass were bonded under a stereoscope.
S1.2 Dielectric Modeling
The direction of DEP force is dependent on the polarity of the real part of Clausius-Mossotti (CM) factor.
Negative DEP is a consequence of negative CM factors, and positive DEP positive CM factors. The frequency at
which the CM factor is zero is the fxo, where cells are invisible to the electric field as cell membrane cannot be
charged to deflect field lines at this frequency. CM factor is dependent on medium conductivity, and as medium
conductivity is increased the real part of CM can take negative values. Assuming cell complex permittivity does
not change with extracellular conductivity largely, if the medium conductivity exceeds a cell type specific value
the polarity of CM factor is negative in the frequency range 1 kHz – 10 MHz. Therefore, in physiological buffers
(~1.5 S/m) cells usually exhibit negative DEP response. Cell complex permittivity can be calculated using the
double shell model, given as [1]:
*
*
 cell
  mem
2(1   1 )  (1  2 1 ) E1
,
(2   1 )  (1   1 ) E1
where subscript mem stands for cell membrane. The factor  1 is given as,
(2)
 1  (1  t / a)3 , where
t is the
membrane thickness, a is the cell radius. The parameter E1 is given as:
*
 cyt
2(1   2 )  (1  2 2 ) E 2
E1 
,
*
 mem (2   2 )  (1   2 ) E 2
where
(3)
 2  (an /( a  t ))3 , and a n is the radius of the nucleus. Subscript cyt stands for cytoplasm. E2 is given
by:
E2 
where
*
 ne
2(1   3 )  (1  2 3 ) E3
,
*
 cyt (2   3 )  (1   3 ) E3
(4)
*
*
 3  (1  tn / an )3 , E3   np
/  ne
, and t n is the nuclear envelope thickness, np and ne subscripts stand
for nucleoplasm and nuclear envelope, respectively.
S1.3 Uncertainty Analysis
The impedance measurement accuracy at the 4294A terminals are ±0.08% [ohms] for impedance magnitude and
±0.0008% [rad] for phase angle for the ranges used in this study. Using these values, upper and lower bounds for
the dielectric parameters were calculated. Next, the CM factors were calculated, and crossover frequencies
approximated for the upper and lower bounds. The percentile error is calculated using these bounds, which
resulted in maximum of 2.55% uncertainty. In a previous publication, we have given the expression for the CM
factor error [2]. Using this expression and assuming 10% error in calculating the volume fraction, we calculated
the uncertainty in estimating the crossover frequency to be negligible. Lastly, the error is affected by the
quantization of the frequency window in the measurements. We have used 801 logarithmically spaced
frequency points, which brings in around 1.06% error in the estimation of the crossover frequency. The total
uncertainty was calculated using root mean square error.
S1.4 Common Low Conductivity Buffers
Types of Low Conductivity Buffers Used in the Literature
Ref
Ingredients
[3]
Sucrose and Dextrose
[4]
1:21 Dilution of culture medium with sucrose/dextrose solution
[5]
1:21 Dilution of culture medium with sucrose/dextrose solution
[6]
Sucrose and Dextrose buffer
[7]
Sucrose and Dextrose buffer supplemented with RPMI
[8]
Sucrose, Dextrose, and BSA
[9]
Sucrose and Dextrose based buffer
[10]
Diluted PBS
[11]
Sucrose and BSA
[12]
Sucrose and dextrose
[13]
PBS diluted with sucrose and dextrose
[14]
Sucrose and dextrose based buffer
[15]
Sucrose and dextrose based buffer
Conductivity [S/m]
0.002
0.056
0.056
0.056
0.02-0.06
0.04
0.03
0.06-0.3
0.00098
0.07
0.057
0.03
0.03
S1.5 Cell Diameter and Trypan Blue Assay
In the modeling procedures, all cells were assumed as perfect spherical particles. This is a reasonable
approximation as cells become nearly spherical after non-spherical adherent cells (Con8) were harvested from
the culture flask by trypsinization. The cell diameter was determined using ImageJ (U.S. National Institutes of
Health, Bethesda, MD, USA) with at least 30 chondrocytes and 50 Jurkats measured at each timepoint for each
condition [16, 17]. A student's t-test was performed on the data sets. The test indicated that while the size
distributions of chondrocytes and Jurkat cells in growth medium are significantly different (p < 0.01 for
chondrocytes and p < 5×10-8 for Jurkats) from those in LCB.
The dead:live ratio of cells was determined by a Trypan Blue exclusion assay (Sigma-Aldrich, St. Louis, MO, USA).
Typan Blue is indicative of membrane poration and is generally used as a marker for dead cells.
S1.6 Intracellular Calcium Imaging
Intracellular calcium imaging was performed on chondrocytes cells using procedures previously described [18].
Cells were cultured on glass coverslips, loaded with Fura-2/AM dye (Sigma-Aldrich), and placed into a vacuum
perfusion chamber mounted on an IX71 microscope (Olympus, Center Valley, PA, USA). Cells were maintined
using a physiological solution consisting of 5.4 mM KCl, 140 mM NaCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM
glucose, and 5 mM HEPES. The cells were recorded in the physiological solution for 60 seconds before LCB was
perfused and washed out with the physiological solution after 3 minutes. Alternating excitation at 340 and 380
nm was performed using a Lambda DG4 switcher (Sutter, Novato, CA, USA), and emission at 510 nm was
recorded with a C9100-02 electron multiplication CCD digital camera (Hamamatsu Photonics, Hamamatsu, JPN).
The intracellular calcium concentration was calculated using a calibration kit (Life Technologies, Carlsbad, CA,
USA) and the equation:
𝑅 − 𝑅𝑚𝑖𝑛
[𝐶𝑎2+ ] = (
) × 𝐾𝐷 × 𝛽
𝑅𝑚𝑎𝑥 − 𝑅
where the recorded ratios are R, zero calcium ratio is Rmin, ratio at calcium saturation is Rmax, the MP
rollseffective dissociation constant is KD, and the ratio of free:bound dye is β [19].
Due to the use of vacuum perfusion for the rapid change of solutions, non-adherent Jurkat cells were unable to
be imaged, as they would be washed away.
S1.7 Reverse Transcription and Real-Time PCR Analysis
Con8 cells were cultured and incubated in LCB for one hour. RNA from control and treated cells were directly
extracted from tissue culture dishes, and genomic DNA eliminated using a Direct-zol™ RNA MiniPrep (Zymo
Research, Irvine, CA, USA). Complimentary DNA (cDNA) was generated using an RT-First Strand Kit (Qiagen,
Valencia, CA, USA). Polymerase chain reactions (PCRs) were performed using SYBR green detection (Qiagen) and
customized ion channel array plates (Qiagen) in a BioRad CFX96 system (BioRad, Hercules, CA, USA). These
customized plates provide gene expression data on 84 different ion channel-related genes. Manufacturer
guidelines were used for PCR reaction volumes and cycle parameters.
S2 Supplementary Figures
Figure S2.1. Picture of the impedance measurement device (a) and schematic of the setup (b).
Time-Dependent Diameter Changes in LCB
20.0
Jurkat
Chondrocytes
Diameter (µm)
**
15.0
***
*
10.0
5.0
GM
0
10
20
30
Time in LCB (minutes)
40
50
60
Figure S2.2. Cell diameter changes of chondrocytes and Jurkats in LCB. Measurements were taken when cells
were in growth media (GM) and then interacting in LCB over the course of an hour. Significance is shown where
* p < .05, ** p < .01, and *** p < 5×10-8.
Figure S2.3. Time evolution of the mean separability parameter of the chondrocyte–Jurkat cell pair in low
conductivity buffer. The conductivity of the buffer is 0.06 S/m. Vertical bars show the standard deviation.
LCB
****
****
****
****
****
****
***
LCBS
0.0540
***
***
****
***
****
***
LCBSN
PBS
****
****
***
***
***
***
****
0.9084
****
***
**
****
***
***
Jurkats
PBSS
*
****
*
****
****
****
***
Recovery
*
****
0.2467
****
****
****
0.0554
Chondrocytes
GM
LCB
LCBS
LCBSN
PBS
PBSS
Recovery
GM
1.0000
**
**
**
**
***
*
Figure S2.4. Statistical comparisons of chondrocytes and Jurkats in different buffer compositions. P-values for
comparisons of chondrocytes (white) and Jurkats (light gray) of growth medium (GM), low conductivity buffers
(LCB), LCB + 10% serum (LCBS), LCBS + 20mM sodium (LCBSN), phosphate buffered serum (PBS), PBS + 10%
serum (PBSS), and a recovery where after 1hr incubation in LCB the measurements were taken after the LCB was
replaced with GM for 1hr. The dark gray is comparison of each buffer formula between chondrocytes and
Jurkats. P-values were calculated using a paired T-Test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;
non-significant values are shown).
Figure S2.5. Relative amount of Typan Blue positive vs. negative cells in chondrocytes and Jurkats. Trypan Blue
counts were done at 10 minute intervals over the course of an hour. Typan Blue is able to penetrate the cell
membrane when large pores are formed. These large pores are commonly found in dead cells.
Figure S2.6. Time evolution of crossover frequency of Jurkat cells suspended in LCB (0.08 S/m, solid line); LCB
supplemented with 10% serum, aminoacids, vitamins (0.18 S/m, dashed line); LCB supplemented with 20 mM
NaCl (0.25 S/m, dots and dashes).
Figure S2.7. Evolution of dielectric nuclear properties of Con8 (solid line) and Jurkat (dashed) cells with time. All
repetitions are presented in the figures.
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