Cell Stretching Measurement Utilizing Viscoelastic Particle Focusing

Nov 19, 2012 - Department of Chemical Engineering, Kwangwoon University, Seoul ... Department of Molecular Science and Technology, Ajou University, ...
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Cell Stretching Measurement Utilizing Viscoelastic Particle Focusing Sukgyun Cha,† Taeho Shin,‡ Sung Sik Lee,§ Wooyoung Shim,∥ Gwang Lee,∥,⊥ Seong Jae Lee,∇ Younghun Kim,*,† and Ju Min Kim*,‡ †

Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea Department of Chemical Engineering, Ajou University, Suwon 443-749, Republic of Korea § Institute of Biochemistry, ETH Zurich, Zurich, CH 8093, Switzerland ∥ Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea ⊥ Institute for Medical Sciences, Ajou University School of Medicine, Suwon 443-749, Republic of Korea ∇ Department of Polymer Engineering, The University of Suwon, Gyeonggi 445-743, Republic of Korea ‡

S Supporting Information *

ABSTRACT: We present an efficient method for measuring cell stretching based on three-dimensional viscoelastic particle focusing. We suspended cells in a biocompatible viscoelastic medium [poly(vinylpyrrolidone) solution in phosphate-buffered saline]. The medium viscoelasticity significantly homogenized the trajectories of cells along the centerline of a simple straight channel, which could not be achieved in conventional Newtonian media. More than 95% of red blood cells (RBCs) were successfully delivered to the stagnation point of a crossslot microchannel and stretched by extensional flow. By computational simulations, we proved that this method prevents inaccuracies due to random lateral distributions of cells and, further, guarantees rotational-free cell stretching along the shear-free channel centerline. As a demonstration, we characterized the differences in RBC deformabilities among various heat treatments. Furthermore, we monitored the decrease of deformability due to nutrient starvation in human mesenchymal stem cells. We envisage that our novel method can be extended to versatile applications such as the detection of pathophysiological evolution in impaired RBCs due to malaria or diabetes and the monitoring of cell quality in stem cell therapeutics.

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stretching of the deformable materials can be significantly affected by the trajectory of each material (or its initial lateral location).10 Therefore, the seeming heterogeneity in the distribution of measured stretching may originate from the nonuniform field kinematics inside the stretching device, which can be confused with the intrinsic properties of the materials. Second, there is a low probability of finding the deformable materials near the stagnation point, where a strong and uniform extensional field is present.7b One possible solution to these problems is to focus the materials along the channel centerline, which would simultaneously homogenize the material trajectories into the cross-slot.3b Recently, Gossett et al. demonstrated that particle focusing using inertial flows in asymmetrically curved channels could successfully homogenize the cell trajectories into the cross-slot, which was incorporated to measure the stretching of various deformable particle such as droplets and cells.3b

ell deformability is a promising label-free biomarker for the diagnosis of health.1 For instance, the deformability of red blood cells (RBCs) in patients suffering from sickle-cell disease, malaria, or diabetes is distinguishable from that of healthy cells.2 In addition, the deformability measurement has been suggested as an efficient platform for the quality control of stem cells.1b Recent microfluidics-based platforms3 for measuring cell deformability have attracted much attention due to their high throughput and potential for automated measurement.1b,c,4 One such promising microfluidics-based approach is the visual measurement of cell stretching in various flow fields using video microscopy.3b,5 Extensional flow fields are attractive for stretching deformable materials such as cells,3b,5 vesicles,6 and DNA.7 In this flow type, affine stretching exponentially increases as the strain experienced by the materials is accumulated,8 and the purely extensional field is rotational-free,9 thus, deformable materials can be highly stretched.9 Microfluidic cross-slots have been used as platforms to generate extensional flow fields at the stagnation point (or the central region).3b,6,7b However, this device presents limitations. First, the extensional field is not uniform inside a practical stretching device, and thus, the © 2012 American Chemical Society

Received: September 23, 2012 Accepted: November 9, 2012 Published: November 19, 2012 10471

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Figure 1. Schematic diagrams for (a) the geometry of the device and (b) a magnified view of the cross-slot region in the device. Numerical simulations for the flow fields in the cross-slot channel with an upper convective Maxwell model at De = 0.1 for (c) the flow-type parameter α, which is purely extensional flow if α → 1 and purely rotational flow if α → −1, and (d) the distribution of the nondimensional strain rate (ε̇ )̂ , where the velocity and length scale were normalized with the average velocity (U) and half the channel height (h/2), respectively. The circles with diameter of 25 μm in panels c and d represent a region where the strain rate is at least 95% of the maximum strain rate at the center. (e) Trajectories of PS beads in a Newtonian medium [24.7 wt % dextran solution in PBS; flow rate (Q) = 160 μL/h (De = 0)]. Trajectories of PS beads in a viscoelastic medium [6.8 wt % PVP solution in PBS; Q = 40 μL/h (f) and 160 μL/h (g) (De = 0.04 and 0.17)] at the cross-slot. For panels e−g, the trajectories were obtained using the z-projection with the “standard deviation” option in the ImageJ software package (NIH).

(PBS)] was used as a suspending medium for the cell stretching measurement. The viscoelasticity of the fluid flow was characterized by the Deborah number [De ≡ λ/Tf ≡ λ(U/ D)], or Weissenberg number (Wi ≡ λε̇c). The Deborah number is the relative ratio of a material time scale to a flow time scale (Tf ≡ D/U), and the material time scale of a polymer solution is the relaxation time, λ.13 U is the average velocity in the channel, and ε̇c is a characteristic strain rate. The Weissenberg number, which is interpreted as the relative ratio of elastic to viscous forces in a viscoelastic flow, is frequently identical to the Deborah number.13 In this work, we set ε̇c to U/D, and thus, Wi is identical to De. The flows in this study were inertialess, and hence, the Reynolds number (Re ≡ hUρ/μ) was assumed to be 0 (the maximum Re throughout this work 95%, RBCs) at the stagnation point of the cross-slot channel and monitored their stretching in the extensional flow. Further, we demonstrated that this novel method could (1) characterize the deformability change of nonspherical RBCs, which experienced various heat shocks, and (2) monitor the decrease of deformability in human mesenchymal stem cells (hMSCs) due to nutrient starvation.

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THEORETICAL BACKGROUND Parts a and b of Figure 1 depict schematic diagrams of the cross-slot used to generate extensional fields in this work. Deformable cells were transported from inlet to outlets as denoted in Figure 1a, and cell stretching was observed near the stagnation (center) of the cross-slot channel (Figure 1b). Both the width (w) and height (h) were constant at 50 μm over the straight regions. A magnified view of the cross-slot region is shown in Figure 1b, for which the sidewall in the first quadrant was designed to follow a hyperbolic function [y = 1875/x (μm) (x ∈ [25 μm, 75 μm])] and the sidewalls of the remaining quadrants are axially symmetric to that of the first quadrant. The origin of the coordinate axes is the center of the cross-slot. In this work, a polymer solution [6.8 wt % poly(vinylpyrrolidone) (PVP) solution in phosphate-buffered saline 10472

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necessitate additional design steps to incorporate Dean flow effect with serpentine channels.3b

Parts c and d of Figure 1 clearly show that the flow fields are highly nonuniform and a uniform α and ε̇ ̂ region exists only near the channel center. This heterogeneity of the flow kinematics implies that the cells will experience different kinematic histories according to the trajectories that are determined by their initial lateral locations, which will deteriorate the accuracy of the cell deformability measurement. In Figure 1c, the central circle with diameter 25 μm corresponds to the region where the strain rate (ε̇ )̂ is at least 95% of the maximum strain rate in Figure 1d (we note that the relative area of the 95% maximum strain rate region to the whole cross-slot is quite similar to that found for a Newtonian fluid7b). As shown in Figure 1c, the flow type is almost purely extensional (i.e., α = 1) inside the circle, and hence, it is expected that the cells experience rotational-free dynamics in this region. On the other hand, the midplane between the two walls is a shear-free plane in the z-direction. Taken together, these numerical results indicate that cells experience shear-free (or rotational-free) stretching only if they move along the channel centerline. The kinematic histories are also homogenized in such circumstances. The viscoelasticity of a polymer solution significantly contributes to the dehomogenization of particle distribution under suspension flow, and Ho and Leal theoretically predicted lateral particle migration by imbalanced first and second normal stress differences in the second-order fluid.16 Leshansky et al.11b demonstrated that the lateral particle migration under viscoelastic flow could be exploited to focus rigid particles on the midplane of a slit microchannel (i.e., two-dimensional particle focusing). In addition, they predicted the particle distributions with their model based on scaling argument, which were in good agreement with the experimental data.11b Some of the authors recently demonstrated that rigid spherical particles were focused along the channel centerline by the synergistic effects of inertial and elastic forces under inertial viscoelastic flow.11c In addition, under inertialess flow, it was demonstrated that cells could be three-dimensionally focused along the channel centerline when the viscoelasticity of a polymer solution was synergistically combined with the deformability-induced wall lift force.11d Meanwhile, it was predicted that the lateral velocity (v) normalized with the streamwise average velocity (u) is proportional to both Wi (≡ De in this work) and the square of the aspect ratio (β) between the particle diameter (a) and the channel height (h), i.e., v/u ∼ (De)(β2) in planar11b and circular11a,17 channels. Later, it was shown that the same scaling law can be also applied to a square channel considered in this work.11d This viscoelasticity-based method was also applied to focus submicrometer-sized rigid particles and the enhancement of DNA focusing.18 In this work, we utilize the viscoelasticity-based particle focusing to homogenize cell trajectories along the channel centerline. It is based on a purely hydrodynamics-based passive method which does not require any active instruments such as an electric field generator. Furthermore, the particle focusing is extremely easy because cells are focused in a self-modulated manner along the shear-f ree centerline of a simple straight channel,11c which has unique merit compared with another promising passive method, i.e., inertial particle focusing.12 In the inertial microfluidics, particles migrate toward equilibrium positions between the channel centerline and walls in a straight channel and particles rotate at the equilibrium positions,19 which

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EXPERIMENTAL SECTION Four-walled poly(dimethylsiloxane) microfluidic channels were fabricated following conventional soft lithography methods20 (cf. Yang et al.11c for the specific conditions). We used a 6.8 wt % PVP (Sigma-Aldrich, MW = 360 000 g/mol) solution in PBS (Sigma-Aldrich) as a viscoelastic fluid (viscosity = 90 cP; relaxation time = 9.4 × 10−4 s).11d The addition of this high molecular weight PVP does not significantly alter the osmolarity of the isotonic solution (PBS), and thus, the ratio of RBC surface area to volume is not changed.4b Thus, it is considered that PVP does not significantly alter cell mechanical properties. The PVP-based solution has been widely used for hematology studies,5,21 and PVP has been also used to slow down sperm mobility prior to intracytoplasmic sperm injection.22 A 24.7 wt % dextran solution (Sigma-Aldrich, MW = 70 000 g/mol) in PBS was used as a Newtonian fluid (viscosity = 91 cP)11d for comparative studies. A surfactant (0.5 wt % Pluronic F-68, Sigma-Aldrich) was added to the PVP mixture to prevent particle−particle adhesion in all experiments. Polystyrene (PS) beads (6 μm diameter) were used as model rigid spherical particles, which were synthesized following a previously reported procedure.11c Whole rat blood, purchased from Orient Bio (Seongnam, Korea), was provided as stored in K2EDTA-treated blood-collection tubes (BD Vacutainer). To measure the effect of heat, whole blood samples were immersed in a water bath for 2 min at four different temperatures (45, 50, 55, and 60 °C) before the blood samples were mixed with the viscoelastic medium (6.8 wt % PVP solution in PBS) at a ratio of 4.4 μL of whole blood to 4 mL of viscoelastic medium. hMSCs were obtained from the iliac crest of normal human donors. This hMSC study was approved by the Scientific− Ethical Review Board of Ajou University Medical Center (AJIRB-CRO-05-126). Briefly, hMSCs were cultured in lowglucose Dulbecco’s modified Eagle’s medium (Invitrogen Corporation, United States) containing 10% fetal bovine serum (Gibco, United States) and 1% penicillin/streptomycin (Gibco, United States). hMSCs were incubated for 24 h in PBS with and without 6.8 wt % PVP solution. In this work, the cell suspensions were flowed using syringe pumps (11 Plus, Harvard Apparatus; KDS 100, KD Scientific). The stretching of cells was observed with a ×20 objective installed on each of two optical microscopes (BX60, Olympus; IX71, Olympus), and the images were acquired using a highspeed CCD camera (MC2, Photron) with 3000 frames/s and a 1/5000 s exposure time. The lengths of cells were measured using ImageJ software (NIH). To assess the viability of hMSCs, we performed an MTS (3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) assay and fluorescence-activated cell sorting (FACS) analysis (annexin V-FITC apoptosis detection kit, Becton Dickinson Biosciences, United States), according to the manufacturers’ protocols. Briefly, for the MTS assay, hMSCs were transferred to 96-well plates at a density of 5 × 104 per well, and fresh medium containing MTS tetrazolium (Promega, United States) was added. After incubation for 2 h at 37 °C, the optical absorbance at 490 nm of each well was measured using an automated plate reader (BioTek Instruments, United States). The absorbance values were averaged after subtracting blank reference values. For 10473

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Figure 2. Ratio of the focused PS beads and RBCs that passed through the circled region in Figure 1, parts c and d, to the total RBCs flowing into the cross-slot (number of samples = 500; the fractions of focused PS beads and RBCs are displayed in black): (a) PS beads in a Newtonian medium, (b) RBCs in a Newtonian medium, (c) PS beads in a viscoelastic medium, and (d) RBCs in a viscoelastic medium.

Figure 3. (a) Typical image of a stretched RBC at a flow rate of 160 μL/h, where the circled region is identical to those in Figure 1, parts c and d. (b) Kinematic histories experienced by the cells when they move along the channel centerline from the inlet to the center, and then from the center to the outlet, where the x-axis corresponds to the off-center distance from the center (“−” and “+” signs denote the distances in the inlet and outlet directions, respectively). The velocity and the strain rate were normalized with the fully developed velocity along the centerline and the maximum strain rate on the channel center, respectively. (c) Elongation index [EI ≡ (L − L̅ 0)/L̅ 0; L, maximum length of a deformed RBC; L̅ 0, average length of nondeformed RBCs] of RBCs according to the locations in the cross-slot microchannel. Each point was obtained by averaging the EIs of 10 samples. (d) Mean EIs according to flow rates (Q = 0.5, 2.5, 5, 10, 20, 40, 80, and 160 μL/h, with De = 0.0005, 0.0025, 0.005, 0.01, 0.02, 0.04, 0.08, and 0.17, respectively; number of samples = 50). The error bars denote standard deviations.

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Figure 4. Effect of various heat treatment conditions on the deformability. (a) Change in EI [≡ (L − L̅ 0)/L̅ 0; L, maximum length of a deformed RBC; L̅ 0, average length of nondeformed RBCs] relative to heat treatment temperature. The immersion duration in the water bath was fixed at 2 min. (b) Effect of immersion duration in the water bath on the change in EI at 45 °C. Fifty samples were used for panels a and b. The data at 25 °C were the same as those in a control experiment with no heat treatment (Figure 3d). The error bars denote standard deviations.

the fractions of focused RBCs (black in the figures) were higher than those of the rigid PS beads in both the Newtonian and viscoelastic fluids, which can be attributed to the deformability of RBCs, i.e., RBC migration toward the centerline was enhanced by the deformation-induced wall lift force.11d Meanwhile, in Figure 2, parts c and d, particle focusing in the viscoelastic medium showed an increased tightness with an increase in the flow rate for both the PS beads and RBCs because the lateral migration speed by the medium viscoelasticity is proportional to Wi (≡ λ(U/D)11a,b,d,17 (De in this study; also see Figure 1, parts f and g). The nonoverlapping RBCs which passed through the circled region (Figure 1c) at 160 μL/h are ∼110 cells/s, which corresponds to the expected throughput of our current method at the flow rate. Next, we demonstrated the measurement of RBC deformability at the cross-slot in the viscoelastic medium. For these measurements, RBCs that had passed through the 95% maximum strain rate region (the circled region in Figure 3a is equivalent to that in Figure 1c) were chosen with high selection probability, as shown in Figure 2d. In Figure 3a, RBCs approach the stagnation point (the channel center) from the inlets (upper and lower channels), pass through the circled region, and then move to the outlets (left- and right-hand sides). Figure S2 in the Supporting Information demonstrates the change in shape of a single cell over its trajectory. Through computational simulations (Figure 3b), we estimated the kinematic histories experienced by the cells according to their trajectories along the channel centerline, where “−” and “+” signs denote the distance in the inlet and outlet directions, respectively, as shown in Supporting Information Figure S3. At the channel center, it was predicted that the cells would experience the maximum strain rate, as shown in Figure 3b, and their velocity would simultaneously be reduced to 0. We then experimentally traced the RBC deformation to investigate the dependence of their deformation on their location. The deformation was assessed by the “elongation index” (EI), defined as (L −L̅ 0)/L̅0, in a similar way to a previous study,23 where L is the maximum length of a deformed RBC and L̅ 0 is the average length of nondeformed RBCs. In Figure 3c, the RBCs were maximally stretched just before escaping from the circled region, as expected from kinematic analysis (Figure 3b). The EI increased as the RBCs approached the stagnation point, but a relatively constant value was retained even after the RBCs left the circled region. Hence, the maximum EI, when RBCs started to escape from the 95% maximum strain rate region, was chosen as the characteristic cell deformability at the

FACS analysis, cells were harvested and washed twice with cold PBS, and then cells were resuspended in 100 μL of 1× binding buffer. Amounts of 5 μL of annexin V-FITC and propidium iodide (PI) were added to the samples in order to discriminate cells under apoptosis and necrosis, respectively, which were incubated for 15 min in the dark. To stop the reactions, 400 μL of 1× binding buffer was added to each tube. Cells were assayed by a FACS machine (Becton Dickinson FACSvantage, United States), and data analysis was performed with BD FACS-Diva software (Becton Dickinson Biosciences, United States).

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RESULTS AND DISCUSSION We first investigated the effect of medium viscoelasticity on the particle distribution in the cross-slot channel. PS beads (mean diameter = 6 μm) were flowed through the channel in order to observe the distributions. The particles at the inlet were randomly distributed for both the Newtonian and viscoelastic media (not shown here). In the Newtonian medium, the particles are evenly distributed in the cross-slot at all flow rates (we show a representative particle distribution at a flow rate of 160 μL/h in Figure 1e). In the viscoelastic medium, however, the particles were significantly focused along the centerline of the channel as shown in Figure 1f (flow rate = 40 μL/h; De = 0.04) and Figure 1g (flow rate = 160 μL/h; De = 0.17). The particles were more tightly focused at the higher flow rate, as predicted by the previous theory11d (see also Supporting Information Movie S1 for a comparison between the Newtonian and viscoelastic fluids at a flow rate of 160 μL/h). We also present distributions of rat RBCs for the Newtonian and viscoelastic fluids in Supporting Information Figure S1 (also see Supporting Information Movie S2), which shows that the multiple equilibrium positions of rigid particles at the centerline and corners were changed to a single pile along the channel centerline. We note that the centerline and corners are equilibrium positions for rigid particles under inertialess viscoelastic flows,11c but the equilibrium positions at the corners are disrupted for deformable cells such as RBCs by the deformability-induced wall lift force.11d We also quantified the efficiency of particle focusing at the center of the cross-slot. As shown in Figure 2, we analyzed the fraction of particles (number of samples = 500) that passed through the circled region (cf. Figure 1c). The particles in the viscoelastic medium (Figure 2, parts c and d) were more tightly focused along the channel centerline than in the Newtonian cases (Figure 2, parts a and b) for both the PS beads and RBCs, which is explained by the medium elastic force. In the figures, 10475

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Figure 5. (a) Deformability change of hMSCs due to nutrient starvation in 6.8 wt % PVP solution in PBS. The deformability indices were measured at a flow rate of 160 μL/h (De = 0.17). The insets correspond to the representative cell shapes after 0 and 24 h in the PVP solution, and the scale bars denote 25 μm. (b) MTS assays of hMSCs after 0 and 24 h of incubation in PVP solution and PBS without PVP. To evaluate the cytotoxicity toward hMSCs, we performed the MTS assay after 24 h of hMSC incubation in the PVP and the PBS without PVP solutions. Data are the means ± SD of three experiments (*p < 0.05, comparison with 0 and 24 h). (c) hMSCs were incubated for 0 and 24 h in PBS solution and then quantified by FACS analysis after staining with annexin V and propidium (PI). The viability of the cells decreased to 73.7% over time (0−24 h).

corresponding flow rate since this value was less sensitive to the location in the downstream direction. In Figure 3d, there was a sharp increase in the EI when the flow rate was ramped from 0 to 10 μL/h, and the EI was almost saturated at 1.3 as the flow rate approached 160 μL/h. Further, we investigated the effect of heat treatment on RBC deformability. Heat treatment is known to irreversibly reduce RBC deformability. When an RBC is exposed to high temperatures, its membrane stiffens due to heat-induced cross-linking reactions among membrane skeletal proteins. It is known that the changes in RBC deformability due to heat occur around 45 °C.24 In Figure 4a, the EIs of the heat-treated RBCs are plotted as a function of heat treatment temperature and flow rate (the data at 25 °C was from a control experiment with no heat treatment). The results demonstrate that the RBC deformability drastically changed above 45 °C for flow rates of 10, 40, and 160 μL/h. In addition, we investigated the effects of duration time at 45 °C and could detect a gradual change in the cell deformability. In Figure 4b, the RBC deformability decreased up to 20 min, but it did not significantly change thereafter. Finally, we applied our method to assess the freshness of hMSCs. In normal stem cell therapeutics, before the cells are injected into the targets, the cells are transferred from a cell culturing medium to PBS.25 Thus, the cells are inevitably starved, which may seriously affect the healthiness of the cells. We compared the deformability and cell diameter of hMSCs, respectively, before and after starvation (24 h) from the culturing medium to the PVP solution in PBS (Figure 5a). In Supporting Information Figure S4, the definitions of deformation index and cell size are denoted. We found that the cells that experienced prolonged starvation (24 h) in the PVP solution were significantly less deformable than fresh cells (0 h). We validated whether PVP affects the viability of the stem cells by MTS. The MTS assay results clearly showed no distinctive difference in viability between the two buffers after 24 h (Figure 5b). In addition, we also performed FACS analysis with annexin V and PI in order to investigate whether the cells experienced apoptosis or necrosis processes over 24 h in PBS (Figure 5c). After incubation in PBS for 24 h, the cells showed

a higher degree of apoptosis and necrosis than control cells. Specifically, the viabilities of the cells were as follows: 0 h, 91.7%; 24 h, 73.7%, and the decrease in cell viability after starvation is consistent with those of the previous report.25 Thus, the FACS data showed that the cells exposed to starvation environments for 24 h in PBS suffered from apoptosis and necrosis, which can be considered the reasons for the decrease in cell deformability after starvation. On the other hand, we speculate that the decrease in cell diameter after starvation, as observed in Figure 5a, results from apoptosis.26 Taken together, we conclude that our method can successfully detect deformability decreases accompanying change in cell viability. The current study demonstrates that our novel method can be utilized as an efficient label-free platform to analyze the freshness of the stem cells, which will be useful in monitoring cell quality.1b

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CONCLUSIONS

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ASSOCIATED CONTENT

We have demonstrated an efficient method for measuring cell deformability based on the extensional field using viscoelastic particle focusing. The method is useful in preventing inaccuracies in the measurement of deformation in extensional flows, which arises from inhomogeneous kinematic histories according to different lateral locations in a Newtonian medium. Our novel deformability measurement method successfully monitored the changes in RBC deformability due to heat. In addition, we showed that our method can be utilized to assess the change in hMSC deformability due to starvation. Finally, we envisage that this medium viscoelasticity-based method will be versatile in the study of various length scales of deformable materialsnot only micrometer-sized cells but also submicrometer-sized particles and DNA.18

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 10476

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(20) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550− 575. (21) (a) Baskurt, O. K.; Boynard, M.; Cokelet, G. C.; Connes, P.; Cooke, B. M.; Forconi, S.; Liao, F. L.; Hardeman, M. R.; Jung, F.; Meiselman, H. J.; Nash, G.; Nemeth, N.; Neu, B.; Sandhagen, B. Y.; Shin, S.; Thurston, G.; Wautier, J. L. Clin. Hemorheol. Microcirc. 2009, 42, 75−97. (b) Dobbe, J. G.; Streekstra, G. J.; Hardeman, M. R.; Ince, C.; Grimbergen, C. A. Cytometry 2002, 50, 313−325. (22) Vansteirteghem, A. C.; Nagy, Z.; Joris, H.; Jiaen, L.; Staessen, C.; Smitz, J.; Wisanto, A.; Devroey, P. Hum. Reprod. 1993, 8, 1061− 1066. (23) Forsyth, A. M.; Wan, J.; Ristenpart, W. D.; Stone, H. A. Microvasc. Res. 2010, 80, 37−43. (24) Mohandas, N.; Clark, M. R.; Jacobs, M. S.; Shohet, S. B. J. Clin. Invest. 1980, 66, 563−573. (25) Lee, K. A.; Shim, W.; Paik, M. J.; Lee, S. C.; Shin, J. Y.; Ahn, Y. H.; Park, K.; Kim, J. H.; Choi, S.; Lee, G. Cytotherapy 2009, 11, 688− 697. (26) Darzynkiewicz, Z.; Juan, G.; Li, X.; Gorczyca, W.; Murakami, T.; Traganos, F. Cytometry 1997, 27, 1−20.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.K.); [email protected] (J.M.K.). Fax: +82-2-941-5769 (Y.K.); +82-31-219-1612 (J.M.K.). Phone: +82-2-940-5768 (Y.K.); +82-31-219-2475 (J.M.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Ki Ryung Choi in the Department of Molecular Science and Technology at Ajou University for MTS and FACS measurements. This research was supported by the Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (Nos. 2010-0003589 and 2010-0007050).

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REFERENCES

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dx.doi.org/10.1021/ac302763n | Anal. Chem. 2012, 84, 10471−10477