Quantitative Investigation for the Dielectrophoretic Effect of Fluorescent Dyes at Single-Cell Resolution.

Most of the microscopy-based, quantitative assays rely on fluorescent dyes. In this study, we investigated the impact of fluorescent dyes on the dielectrophoretic response of the mammalian cells. The dielectrophoretic measurements were performed to quantify whether the fluorescent dyes alter the dielectrophoretic properties of the cells at single-cell resolution. Our results present that when 10 Vpp electric field is applied, the fluorescent-labeled cells experienced the crossover frequency at 8-10 kHz, whereas the label-free cells exhibited at 16-18 kHz.

Introduction

In the late 19th century, the discovery of organic fluorescent compounds provided an avenue for capturing the dynamic processes of living organisms. Various fluorescent dyes have been developed. They have become essential tools for powerful techniques ranging from live-cell fluorescence imaging to flow cytometry, with various applications including the detection of substances, the tracking of single molecules, and the visualization of post-translational modifications.[1−7] Although label-free assays that exclude phenotypic and genetic modifications might be more suitable for many life sciences and medical applications, fluorescent dyes remain one of the most valuable tools for current state-of-the-art assays and technologies. Various fluorescent dyes have been developed with improved sensitivity, selectivity, specificity, detection speed, repeatability, photostability, brightness, and biocompatibility.[1,2,8,9] Modern biotechnological tools in conjunction with these better-quality fluorescent dyes will play a significant role in understanding cellular and subcellular dynamics in the near future as well.

This study presents the dielectrophoretic behavior of pre- and postlabeled single cells when stained with commercially available fluorescent dyes. Dielectrophoresis (DEP) is one of the label-free characterization methods that directly and quantitatively determine whether or not fluorescent dyes alter the intrinsic properties of the cells. Herbert Pohl introduced the DEP phenomenon in the 1950s as the motion of an electrically polarizable particle in a nonuniform electric field.[10,11] The dielectrophoretic force depends on the permittivity of the suspending medium of the cells (εm), the radius of the cell (r), the real part of the Clausius–Mossotti factor (fCM), and the applied electric field (E), as represented in eq .

The Clausius–Mossotti factor is defined as in eq , where εc* is the complex permittivity of the cell, and εm* is the complex permittivity of the medium.

The complex permittivity (ε*) depends on permittivity (ε), conductivity (σ) of the cell/medium, and frequency (f) of the electric field (E), as expressed in eq , where j shows the imaginary number .

When Re[fCM] > 0, the strong electric field regions attract the cells, and they are influenced by “positive DEP (pDEP)”; when the cells are repelled from the high field strength, they are designated “negative DEP (nDEP)”. Both the pDEP and nDEP behavior of the cells rely on their polarizability difference with respect to their surrounding medium. When the polarizability difference between the cells and their surrounding medium is almost negligible, the DEP forces become very weak, to almost zero. This specific frequency is known as “crossover frequency”, which determines the dielectrophoretic characteristics of the cells.[12−15]

Since the 1950s, many new DEP tools and methods with improved sensitivity, throughput, and practical usage have been developed.[16−20] Here, we used the three-dimensional (3D) carbon-electrode DEP, which was introduced by Dr. Martinez-Duarte and his colleagues. It has been previously used in a variety of cell separation applications including bacteria, yeast, and mammalian cells.[13−15,21]

In this study, we investigated the dielectrophoretic effect of two different commercially available membrane-permeant reactive tracer dyes (The CellTracker Green CMFDA (5-chloromethyl fluorescein diacetate) and CellTracker Red CMTPX (Fisher Scientific) on U937, the (pro-) monocytic, human myeloid leukemia cell line (ATCC CRL 1593.2). As these two dyes are well-suited and widely studied not only for being easy to use but also adequate for monitoring the behavior of cells, cell location or movement, and long-term cell tracking thanks to retaining in the living cells through several generations. Besides, multiplexing green and red fluorescent dyes allows the observation of cell–cell communication and protein expressions without cross-contamination. Although these dyes were studied for their cytotoxicity, stability, and other chemical properties, according to our knowledge, investigation on the dielectrophoretic properties of these commercially available fluorescent cell tracker dyes have not been performed yet.[22,23]

Results and Discussion

U937 cells were cultured in an RPMI (Rosewell Park Memorial Institute, Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% Pen/Strep (penicillin–streptomycin, Sigma-Aldrich) in 75 cm2 flasks (Corning T-75 flasks) in an incubator (NUVE EC160), in which 37 °C, 5% CO2, and humidity were maintained. The CellTracker Green CMFDA and CellTracker Red CMTPX dyes were used to prepare a 1 μM staining solution in fresh RPMI medium. Complementing the fluorescent dyes into the medium did not change the conductivity of the medium. The number of U937 monocyte cells was adjusted to 1 × 105 cells/mL using a hemocytometer (Marienfeld). Next, the cells were cultured in the staining solution in the incubator for 30 min. Then, the stained cells were harvested at 3000 rpm (Hettich EBA 20 centrifuge) for 5 min to remove any residual dye in the culture media. Afterward, the cells were resuspended in a low-conductive DEP buffer twice. The DEP buffer was prepared using 8.6% sucrose (BioFroxx), 0.3% glucose (Sigma-Aldrich), and 0.1% bovine serum albumin (PAN Biotech) in deionized (DI) water. The conductivity of the DEP buffer was measured as 20 μS/cm using a conductivity meter (Corning, 311 conductivity).

The dielectrophoretic setup consists of a function generator, an upright, optical microscope integrated with a camera, a computer, a syringe pump, and the 3D carbon-DEP device. There are two 20–200 μL pipette tips at the inlet and outlet of the electrode-array microchannel to create reservoirs. The Tygon tubing connects the syringe and the microchannel in the system.

The dielectrophoretic characterization of label-free and stained U937 monocyte cells was performed using the experimental setup illustrated in Figure . First, the 3D carbon-DEP chip was sterilized by flowing 70% ethanol and then DI water prior to the experiments. Next, the bubbles inside the microchannel were removed and the chip was filled with the low-conductive DEP buffer. The cells were prepared as explained above and introduced into the chip with a 10 μL/min flow rate using the syringe pump (New Era Pump Systems, Inc., NE-1000). When the cells reached the region of the carbon electrodes, the flow was stopped. The cells were settled when a signal with 10 Vpp at frequencies between 1 kHz and 20 MHz was applied from the function generator (INSTEK—GFG-8216A). After the DEP exposure, the cells were collected from the device into a collection tube for further inspection. A Nikon Eclipse, an upright optical microscope with 10× objective, was used to capture videos with 1 frame/s frame rate during the experiments (Figure ).

Figure 1

Illustration of the experimental setup.

Figure 2

Image of the label-free U937 cells in the 3D carbon electrode array. Green arrows show the cells, black circles are the carbon electrodes, and black lines are the connection wires of the electrodes.

The obtained videos were analyzed as demonstrated in Figure and detailed in the previous studies of Dr. Martinez-Duarte and co-workers.[24−26] The location of the cells at each frame was rated as strong nDEP (−3), nDEP (−2), weak nDEP (−1), crossover (0), weak pDEP (1), pDEP (2), or strong pDEP (3) according to their positions with respect to the carbon electrodes.

Figure 3

Schematic diagram for the image analysis on the 3D carbon-electrode microchip that represents the DEP regions, according to the captured image in Figure .

Figures and 5 show the obtained dielectrophoretic behavior of the label-free U937 monocytes. The monocytes were stained with CellTracker Red and CellTracker Green dyes. To delineate whether the applied fluorescent dyes affect the DEP characteristics of the cells, dielectrophoretic forces were applied between 1 kHz and 20 MHz at 10 Vpp in the 3D carbon-DEP chip. As there was no drag force because of the fluid flow in the system, the translational movement of the cells was due to the exhibited DEP forces (eq ). Next, the location of the cells was correlated with their DEP responses. The position of each cell was traced in each frame using the obtained movies (Supporting Movie). ImageJ was used to analyze the captured images. The mean value of the positions of the cells with standard deviations at each frequency was calculated using the Prism software (GraphPad).

Figure 4

DEP response of monocytes stained with CellTracker Red (white circles), CellTracker Green (black circles), and DEP response of unlabeled monocytes (star). A total of 50 cells were tracked from 1 to 30 kHz at 10 Vpp.

Figure 5

DEP responses of monocytes at single-cell resolution. (a) Unlabeled monocytes, (b) monocytes stained with CellTracker Red, (c) monocytes stained with CellTracker Green. Fifty cells were tracked for each frequency from 1 to 30 kHz at 10 Vpp. The red lines show the mean and standard deviation for the cells at each frequency.

Conclusions

Fluorescent dyes have been widely used as tremendous tools for cell labeling in a wide variety of applications of life sciences and medicine, such as monitoring chemotaxis and invasion, tracking cell movement and migration, and quantifying proliferation. They are easy to use and compatible with several assays. Their fluorescent signal is retained in living cells through several generations. For these dyes, most of the conventional characterization assays and methods quantify their sensitivity, selectivity, brightness, photostability, specificity, toxicity, and photochemical properties. As most of the fluorescent dyes are capable of penetrating through the cell membrane, passing into the cytoplasm, and being impermeant, we investigated whether the fluorescent dyes change the permittivity and conductivity of the cells that may influence the dielectric properties of the cells. To the best of our knowledge, this is the first study that investigates the dielectrophoretic properties of commercially available fluorescent cell tracker dyes. As shown in Figures and 5, there is a slight shift in the crossover frequencies of the cells when they are labeled with membrane-permeant reactive tracers. This minor variation might be negligible for many applications; nevertheless, it might be significant for the dielectrophoretic separation of cells that exhibit very close dielectrophoretic responses at single-cell resolution.