Observing Single Cell NF-κB Dynamics under StimulantConcentration GradientYousef Awwad,† Tao Geng,‡ Albert S. Baldwin,§ and Chang Lu*,†,¶

†School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University, Blacksburg, Virginia 24061, United States‡Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, United States§Lineberger Comprehensive Cancer Center, Department of Biology, School of Medicine, University of North Carolina, Chapel Hill,North Carolina 27599, United States¶Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States

*S Supporting Information

ABSTRACT: Study of cell signaling often requires examination of the cellulardynamics under variation in the stimulant concentration. Such variation hastypically been conducted by dispensing cell populations in a number ofchambers or wells containing discrete concentrations. Such practice adds to thecomplexity associated with experimental or device design and requiressubstantial labor for implementation. Furthermore, there is also potential riskof missing important results due to the often arbitrary selection of discreteconcentration values for testing. In this Letter, we study NF-κB activation andtranslocation at the single cell level using a microfluidic device that generatescontinuously varying concentration gradient. We use only three device settingsto cover stimulant (interleukin-1β) concentrations of 4 orders of magnitude(0.001−10 ng/mL). Such device allows us to study temporal dynamics of NF-κBin single cells under different stimulant concentrations by real-time imaging.Interestingly, our results reveal that, while the percent of cells with NF-κB translocation decreases with lower stimulantconcentration in the range of 0.1−0.001 ng/mL, the response time of such translocation remains constant, reflected by the singlecell data.

There has been increasing evidence that cell signalingprocesses are stochastic in nature and cellular responses to

extracellular stimuli are best studied at the single cell level. Forexample, when cells show an all-or-none response to aparticular stimulus (bistability), only a subpopulation of cellsrespond to the signal.1−5 Conventional bulk measurementsonly yield the lowered population average and do not reveal thesubpopulations. In addition, observation of cellular dynamicswith distinct temporal patterns is impossible with population-level approaches, due to the lack of synchronization amongcells.6

NF-κB is a prime example for stochastic signalingdynamics.7,8 NF-κB is a family of dimeric transcription factorsthat control the expression of hundreds of genes involved incellular stress responses, cell division, apoptosis, and inflam-mation.9−11 In the canonical activation pathway, NF-κB residesin the cytoplasm of the cell (due to the negative regulation ofIκB kinase that is bound to NF-κB heterodimers) untilactivation in response to a wide range of extracellular stimuli(e.g., tumor-necrosis factor-α (TNFα), interleukin-1 (IL-1),lipopolysaccharide (LPS), and CD40 ligand (CD40L)). Signalsare transduced to the IκB kinase (IKKβ) complex. IKKβ thenphosphorylates IκB at two conserved serines, leading to itsdegradation via ubiquitination and proteolysis. This allowslibrated NF-κB (RELA-p50 or c-REL-p50) to translocate to the

nucleus, bind to DNA, and regulate gene transcription within.NF-κB signaling presents rich dynamics at the single cell level.For example, NF-κB responds to endotoxin LPS stimulationwith heterogeneity in the population (“transient and persistentresponders”).5 Furthermore, NF-κB activation triggered byTNF-α and T cell receptor (TCR) appear to be digital (switch-like, as opposed to be analog or graded) with stochasticelement involved in the process.3,12 Not surprisingly, it wasfound that TNF-α (the stimulant) concentration was a criticalparameter that affected NF-κB dynamics.3,13,14

In this study, we use a microfluidic device that allowsexposure of cell culture to a continuous stimulant (IL-1β)concentration gradient. IL-1β is a multifunctional cytokineproduced primarily by activated monocytes/macrophages andby a variety of other cell types. IL-1β plays an integral role inthe immuno-inflammatory response of the body to a variety ofstimuli including infection, trauma, and other bodily injuries.IL-1β activates NF-κB via a pathway similar to that of TNF-α,due to the largely overlapping biological activities of the twocytokines.15 The concentration gradient device allows us to

Received: December 3, 2011Accepted: January 13, 2012Published: January 13, 2012

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investigate concentrations spanning several orders of magnitudewith very few experiments. The simple fluid mechanicsassociated with laminar flows permits accurate computationof the stimulant concentration distribution on the basis of thephysical properties of the stimulant molecule and theoperational conditions. By combining the stimulant concen-tration information with the single cell NF-κB dynamics (i.e.,nuclear translocation) observed by real-time imaging, weestablish the patterns of NF-κB response to IL-1β of variousconcentrations. Other than confirming that the percentage ofresponding cells decreases with IL-1β concentration, moreinterestingly, we discover that the correlation between theresponse time (the duration from the start of the stimulation tothe nuclear translocation) and IL-1β concentration disappearswhen IL-1β concentration is low (0.001−0.1 ng/mL), revealingan upper bound for the response time.Previous studies on stimulant concentration effect applied

chambers or wells with discrete concentrations.3,13,14,16 These

studies required using a large number of units for variousconcentrations, and there was potential risk for not covering theimportant concentration values. In comparison, we use amicrofluidic device with a network of channels for generatingconcentration gradient by controlled diffusive mixing.17−19 Weuse three settings (i.e., inlet concentrations) to cover IL-1βconcentrations that continuously span 4 orders of magnitude(0.001−10 ng/mL). As shown in Figure 1, with the stimulant(IL-1β) solution coming in from one inlet and buffer in fromthe other inlet, the branched array of channels repeatedly splitsand combines microscale streams to result in multiple streamswith different stimulant concentrations that flow into adownstream microfluidic chamber. This generates a stableconcentration gradient that is perpendicular to the flowdirection and maintained over time at steady state. We culturea monolayer of Chinese hamster ovary (CHO)/GFP-NFκBp65cells (Panomics), created by cotransfection of an expressionvector for a fusion protein of turboGFP (Evrogen) and human

Figure 1. Design of the microfluidic concentration gradient device for studying single cell NF-κB dynamics. CHO/GFP-NFκBp65 cells are seeded inone monolayer in the microfluidic culture chamber (having a depth of 60 μm, a width of 0.9 mm, and a length of 11 mm). For stimulation of cells,IL-1β of a specific concentration (0.1, 1, or 10 ng/mL) flows into the device at 0.05 μL/min from the left inlet while neat PBS buffer flows in fromthe right inlet at the same flow rate. The concentration profiles along the width of the culture chamber can be modeled by COMSOL, and theconcentration profiles at Z = 1 and 11 mm are shown in the upper inset graph. The lower inset images show the fluorescence images of the cells afterstimulation by IL-1β (inlet concentration of 1 ng/mL) for 0, 65, 130 min. The images show that at the left side of the culture chamber with high IL-1β concentration, the majority of the cells experience NF-κB translocation. In comparison, at the right side, few cells show translocation.

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NF-κBp65, as well as pHyg into CHO cells, in the microfluidicchamber.20 Cells are flowed into the chamber for seeding fromthe outlet, and there is a microfluidic valve that is half-closedduring cell seeding for preventing cell entry into the channelnetwork. The inset plots show that IL-1β concentration profilealong the width of the culture chamber at any specific locationalong the stream can be modeled using COMSOL (ignoringthe effects of the seeded cells on the flow). The modeled valuesmatch experimental results well as shown by the example oflabeled β-casein (Figure S1 in Supporting Information). At Z =

1 mm (i.e., 1 mm from where the channel network interfaceswith the culture chamber), the maximal IL-1β concentrationalong the chamber width (at the very left) is the same as itsinlet concentration, and the minimal concentration (at the veryright) is 0. In comparison, the maximal concentration becomes93% of the inlet concentration, and the minimum is 8% in thedownstream at Z = 11 mm. As shown in the inset image seriesand Movie S1 in Supporting Information, with inlet IL-1βconcentration being 1 ng/mL, the cells at the left side of theculture chamber present higher percentage of activation (NF-

Figure 2. Variations of the percent cell activation and the response time of single cells with IL-1β concentration. The percentage of activated cells(with NF-κB translocation) varies with different IL-1β concentrations when the device has IL-1β inlet concentrations at (a) 10, (b) 1, and (c) 0.1ng/mL. The data from (a−c) are also compiled in (d) with the concentrations in a logarithmic scale. Each data point in (a−d) is generated by threeseparate trials. The response time (the time from application of IL-1β stimulation to NF-κB translocation) of single cells is plotted against theconcentration that the particular cell is exposed to, when the device has IL-1β inlet concentrations at (e) 10, (f) 1, and (g) 0.1 ng/mL. The data from(e−g) are also compiled in (h) with the concentrations in a logarithmic scale.

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κB nuclear translocation) and faster response than the ones atthe right, due to the concentration gradient.There are two types of measurements we conduct using the

gradient generation device. First, we examine the percentactivation of CHO cells at various IL-1β concentrations. In thiscase, we divide the observation frame (900 μm × 685 μm in thesize) into 9 or 10 vertical slabs with each slab roughly coveringa concentration range of equal size. The IL-1β concentration atthe center of the slab is used to represent the concentration ofthe slab. The population of cells within each slab (N ∼ 30−50cells) was observed for NF-κB translocation during a period of3 h under constant stimulation by IL-1β. The cells undergoingNF-κB translocation (indicated by movement of thefluorescence intensity center from the cytosol to the nucleus)are enumerated, and the percent activation is calculated bydividing the number of activated cells by the total cell number.Second, we also examine the response time for every single cellin the observation frame. The response time is determined byhow long it takes for NF-κB translocation to occur since thestart of the IL-1β stimulation. We are able to determine the IL-1β concentration that each single cell is exposed to, on the basisof the cell location using COMSOL modeling, and correlate itto the response time for the particular cell.Figure 2a−c shows how the percent cell activation varies with

the IL-1β concentration, examined by having the inlet IL-1βconcentrations at 10, 1, and 0.1 ng/mL, respectively. We findthat the percentage of activated cells (exhibiting NF-κBtranslocation) does not vary in the range of 1−10 ng/mL,due to the saturation of cell surface receptors by the stimulant.The percentage of activated cells increases with higher IL-1βconcentration in the range of 0.001−1 ng/mL. Such trend canalso be seen clearly in the Figure 2d with all the data compiledand IL-1β concentration shown in a logarithmic scale. Figure2e−g shows single cell dynamics in the NF-κB signalingdependent on the stimulant concentration. Each marker in thefigures represents correlation between the response time andthe IL-1β concentration, generated by analyzing a single cell.Figure 2e shows that there is no strong correlation between theIL-1β concentration and the response time when the IL-1βconcentration is in the range of 5−10 ng/mL. Then, theresponse time decreased with higher IL-1β concentration in therange of 0.1−5 ng/mL (Figure 2e,f). However, the responsetime becomes independent of the IL-1β concentration again inthe range of 0.001−0.1 ng/mL (Figure 2g). We did not observeNF-κB nuclear oscillations3,21 (i.e., in and out of the nucleus)within the observation time (∼3 h). Since the expression levelof NF-κB fused with the fluorescent protein marker is known tostrongly affect the dynamics and oscillations,22,23 such lack ofnuclear oscillation is possibly due to the characteristics of theCHO cell line used.Our observation of NF-κB activation and translocation yields

interesting insights. Although it is known that the lowerstimulant concentration induces a lower percentage of cells thatrespond with NF-κB activation, it is surprising to reveal that theresponse time of these responding cells does not depend on IL-1β concentration in the very low concentration regime. Ourresults show that while the percentage of activated cellsexperiences a significant and continuous decline from 40% to5% as IL-1β concentration varies from 0.1 to 0.005 ng/mL(Figure 2c), there is no significant change in the response timeat the single cell level in the same concentration range (R2 =0.0372 in Figure 2g). This finding is consistent with theprevious bulk measurements revealing that lower TNFα dose

reduces the amount of nuclear NF-κB while maintaining thesame temporal profile regardless of the dose.13

Microfluidic concentration gradient device provides a simpleplatform for studying NF-κB activity and cell signaling ingeneral at the single cell level. Stimulant concentrationsspanning multiple orders of magnitude can be easily coveredby a small set of experiments. Single cells in a monolayer thatare treated by continuously varying stimulant concentration canbe observed by real-time imaging for their dynamics. Becausewe are able to treat each single cell as an individualexperimental subject, the results yield high statistical signifi-cance. We expect increased application of this approach forsingle cell signaling studies in the future.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information including experimental section, supple-mental data, and video. This material is available free of chargevia the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: changlu@vt.edu.

■ ACKNOWLEDGMENTSThis work was supported by NSF grants CBET 1016547 and0967069 (to C.L.) and NIH AI35098 (to A.S.B.).

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