Correlative Analyses of Nitric Oxide Generation Rates and Nitric Oxide

Jul 24, 2012 - Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, a...
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Correlative Analyses of Nitric Oxide Generation Rates and Nitric Oxide Synthase Levels in Individual Cells Using a Modular CellRetaining Device Yana Shafran,† Naomi Zurgil,† Elena Afrimzon,† Yishay Tauber,† Maria Sobolev,† Asher Shainberg,‡ and Mordechai Deutsch*,† †

The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome, and ‡The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, 52900 Israel S Supporting Information *

ABSTRACT: Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

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Upon activation, human macrophages can express iNOS, mRNA, and protein but produce low NO levels which are difficult to detect.10,11 Therefore, NO detection in human macrophages is mainly performed by bulk measurements of secreted molecules using either chemiluminescence, Griess reaction, or electron paramagnetic resonance spectroscopy.12 Other approaches have attempted to determine intracellular NO by analyzing the distribution of its synthesizing enzymes, NOS. NOSs are dimeric enzymes that catalyze formation of NO and citrulline from arginine and molecular oxygen using heme, biopterin, and NADPH as cofactors. Three isoforms have been identified: endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed and play important roles in blood pressure regulation and neurotransmission, respectively, while inducible NOS (iNOS) is synthesized upon exposure to inflammatory and immunologic stimuli and has been implicated in pathogenesis of numerous diseases. However, the NOS approach is limited because measurement of NOS expression provides only an estimate of cell potential to produce NO. Moreover, an alternative pathway for NO

itric oxide (NO) has been recognized in recent years as one of the major agents in the immune system.1 It is involved in pathogenesis and control of infectious diseases, tumors, autoimmune processes, and chronic degenerative diseases.2−4 Stimulation of endogenous NO synthesis or administration of NO donors has proven to be therapeutically effective in various models of diseases.7,26 Conversely, inhibition of NOS may have therapeutic potential for treatment of diseases mediated by overproduction of NO.27 However, such NObased treatments require cell-based evaluation of NO production rate and levels of its synthesizing enzymes. Despite the major impact of NO on human health, and on the immune system in particular, its short half-life and highly reactive nature complicate determination of its local availability in live cells, and its quantification remains difficult, requiring special analytical approaches.5 NO is released by various types of immune cells including monocytes/macrophages,2−4,6,7 key mediators of the immune system. In these cells, NO is a key component of antimicrobial and tumoricidal immune response.8 NO not only serves as a cytotoxic molecule, producing cell demise along an apoptotic or necrotic pathway, but also gains attention as a regulator of immune function.2,9 © 2012 American Chemical Society

Received: November 4, 2011 Accepted: July 24, 2012 Published: July 24, 2012 7315

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generation in mammals was recently discovered,13 in which the inorganic anions, nitrate and nitrite, are reduced in vivo, to form NO and other reactive nitrogen species (RNS). Therefore, it is clear that, in addition to estimation of enzyme expression, the actual amount of NO and its production rates must be determined. In recent years, it has become clear that due, to the heterogeneity between individual cells within the measured population with regard to the number of NOS positive cells, and the rates of both enzyme expression and activity, the results of bulk assays or pooled samples do not accurately reflect NO level, and there is a need to detect NO in single cells.14 Estimation of NO concentrations in individual cells can be achieved by fluorescence imaging. Although intracellular imaging of NO production by fluorescence indicators provides high temporal and spatial resolution, it is used mainly in end point studies due to the inability to trace the same cells over time. NO is a local mediator, its influence extending only to 100 μm of its origin, and thus must be rapidly synthesized in response to stimuli. In addition, it does not require complex metabolism for clearance, since it is simply diluted and oxidized to nitrite and nitrate as it diffuses away from its source.15 Therefore, it is vital for the study of NO effects, to analyze the short-term dynamics of both generation of NO and expression of its synthesizing enzymes. This can be achieved by continuously monitoring single cells within a population, over varying periods of time. For nonadherent cells like monocytes/ macrophages, however, technical limitations preclude such studies where spatial location of the cell must be controlled during experimentation. Previous studies endeavor to establish practical experimental conditions for analysis of NO produced by different types of activated macrophages5 and stimulators.16 Although these conditions may be most favorable for NO assessment, they are not necessarily optimal physiological situations for analyzing basal level and production rate in nonstimulated cells. We have previously introduced17 a miniature cell-retaining methodology for high content analysis of nonadherent cells. In the current study, the microarray technology has been further expanded to develop a modular cell-retaining device (ModCR) which facilitates successive live and postfixation measurements of the same individual cells. These unique features of the ModCR were used for correlative analysis of NO generation rates and NOS levels in individual cells utilizing fluorescent imaging followed by immunohistochemistry (IHC).

depth), each designed to contain a single untethered cell. The glass microarray is attached to a carrying standard microscope slide (1 mm thick) using UV adhesive. A detailed technical description of the microarray and its fabrication process has been previously presented.17 The flow channel fabrication process is a modification of that previously described.17 It is based on simple stacking of cut-toshape thin layers on top of the microscope slide (Figure 1).

Figure 1. Construction of the ModCR, cell loading, and operation. (a) Fabrication of the ModCR. The ModCR is assembled from two major functional parts. The first is a glass microarray permanently attached to a standard microscope slide. The second is a chamber and flow system/maintaining and manipulating system made by stacking cut-toshape thin layers on top of the microscope slide. (b) The latter part can easily be separated from the glass slide to facilitate the IHC process. At the outset of experiment, the liner is removed, a stopper is placed to prevent the cover from closing, and the cell suspension is loaded into the chamber. The stopper is then removed and the cover is closed, adhering to the flow channel spacer. Capillary action occurs within the flow conduit, between the silicon layer and the cover, enabling addition of fluids which pass through the cell chamber and accumulate in the reservoir. (c) Disassembly of the ModCR is done by gently peeling off all the layers as one, leaving only the microarray attached to the microscope slide.

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MATERIALS AND METHODS Please see the Supporting Information (p S-2) for discussion on materials. Fabrication of ModCR for Successive Live and Postfixation Measurements. The ModCR integrates technical achievements of glass microarray production17 and a cell chamber with flow channel18 into a single, easy to use, modular device suitable for live cell imaging, followed by immunohistochemical staining. It is assembled from two distinct parts: a standard microscope slide with glass microarray, and a removable flow channel. The microarray consists of a small piece of BK7 glass (0.5 mm thick, 4 mm × 4 mm) patterned in its middle with a 2 mm × 2 mm densely packed two-dimensional (2-D) arrangement of hexagonal picoliter wells (picowells) (20 μm diameter, 8 μm

The first layer is a 0.28 mm plastic sheet attached to the microscope slide using a double-sided 0.16 mm adhesive tape. These, together with another identical double-sided adhesive, serve as a spacer reaching up to the level of the microarray. A hole (5.7 mm diameter) is drilled through the entire layer to accommodate the microarray. The second layer, made of a silicon sheet (0.8 mm thick), is placed on top of the upper double-sided adhesive of the first layer. A hole (2 mm diameter) drilled through the silicone sheet in the location of the patterned array creates a cavity which serves as the cell chamber. 7316

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then washed and resuspended in PBS at 2 × 106 cells/mL and immediately introduced into the ModCR for optical measurements. For concomitant analysis of NO and GSH, cells were incubated with DAF-2DA (10 μM) and ThiolTracker Violet (20 μM) under the same conditions, then washed and measured as described above. IHC Staining of NOS. Upon completion of vital measurements, cells were fixed within ModCR by adding 4% formaldehyde for 10 min at room temperature (RT) and incubating in cold 50% methanol at RT for 10 min. Then, the upper part of the device was removed and cells were permeabilized for 5 min at RT with 0.1% saponin in PBS, washed with PBS, and incubated with human blocking IgG (1 μg/10 μL) in PBS for 15 min at RT. Slides with fixed cells were washed again and incubated in PBS containing anti-NOS2 mAb for 6 days at 4 °C (dilution 1:200), and then with a second Cy5-conjugated antimouse IgG at a working titer 1:200 for 30 min at RT. The optical system is described and image analysis discussed in the Supporting Information (p S-2). Statistical Analysis. For each experiment, two to three ModCR devices were used. For each device, a total of four images (one each from four different representative areas) were acquired (∼250−900 individual cells). The mean and SD for each measured parameter were calculated for each device, and data is presented as mean fluorescence intensity (FI) ± SD of all pixels within the cell area for individual cells, or averaged FI ± SD for cell populations. Comparison between cell groups was carried out using a Student’s t test with statistical significance set at p < 0.05. Single-factor analysis of variance (ANOVA) test was used for estimating the stability of intracellular FI variances.

The next layer features the flow channel, a two-ply, doublesided adhesive tape (0.16 mm thick), with an interior edge cut in the shape of a bottle having an open narrow neck (4 mm wide) which stretches above the cell chamber and then gradually widens, creating the waste reservoir. The cover, made of flexible, transparent polycarbonate film (0.175 mm thick) with a 4 mm wide slit, for introduction of liquid aliquots during experimentation, is anchored at the bottom of the waste reservoir creating a flap which can be opened. A small pinhole is punched near the end of the reservoir for air release during the flow of fluids. A removable nonadhesive layer separates the flow channel from the cover to keep them from adhering prior to filling the cell chamber. ModCR Cell Loading and Operation. Cell loading is carried out when the ModCR cover is open and cell chamber exposed (Figure 1b). Cell suspension (7−10 μL, 2 × 106 cell/ mL) is loaded into the chamber, and cells are allowed to settle for 5 min, whereupon the separating layer is removed and the cover is closed by attaching to the adhesive layer. Aliquots of 10−20 μL of media or soluble reagents are added to the slit and spontaneously drawn in by capillary forces. Since individual cells within the picowells share a common space, the entire cell sample is exposed to the same concentrations of solutes. Validation tests for assessing quality of the solution transfer and uniformity of reagent concentrations within the array were previously reported.17,18 Disassembly of the ModCR is achieved by gently peeling off all the layers as one, detaching the adhesive tape from the microscope slide, leaving only the microarray attached to the microscope slide. Cell Growth and Differentiation. U937 human promonocyte cells were maintained in RPMI-1640 medium supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2% glutamine, 2% sodium pyruvate, and 2% HEPES. Cells were maintained in completely humidified air with 5% CO2 at 37 °C. Before use, exponentially growing cells were collected, washed, and resuspended at appropriate concentrations. For induction of cell differentiation, nondifferentiated U937 promonocyte cells were incubated in tissue culture flasks for 48 h in complete medium with 1 ng/mL phorbol myristic acetate (PMA) in completely humidified air with 5% CO2 at 37 °C. On-Array Cell Activation. Nondifferentiated and PMAdifferentiated U937 cell populations were activated within the ModCR. Following probe loading, cells were introduced into the ModCR and a first image was acquired. Cells were activated by adding 10 μL of stimulant solution [lipopolysaccharide (LPS), PMA, or lysophosphatidylcholine (LPC) in final concentration of 1 μg/mL, 2 μg/mL, or 20 μM in phosphate-buffered saline (PBS), respectively] to the slit from which the solution was drawn to the cells within the device. For control, stimulation was induced either by solvent alone [dimethyl sulfoxide (DMSO) or PBS] or by simultaneously adding the stimulants in the presence of Nω-nitro-L-arginine methyl ester (L-NAME) (10 μM). Exposure of cells within the ModCR to exogenous NO was carried out by adding 10−20 μL of NO donor solution DiethylenetriamineNONOate ((Z)-1-[2-aminoethyl)-N-(2ammonioethyl)amino]diazen-1-ium-1,2-diolate) (DETA/NO) (0.01−1 mM) and incubating for the appropriate time. Measurement of Intracellular NO and Reduced Glutathione (GSH). For intracellular NO measurements, cells were incubated with 4,5-diaminofluorescein diacetate (DAF-2DA) (10 μM) for 15 min at 37 °C and 5% CO2,

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RESULTS AND DISCUSSION Estimations of Intracellular NO Levels. U937 cells exhibit monocyte-like characteristics, and differentiate into macrophages by treatment with the tumor promoter PMA,31−33 as demonstrated in morphological changes and in their capability to be stimulated with LPS and γ-interferon.19 For the assessment of the basal NO generation rate, nondifferentiated and PMA-differentiated U937 cells were loaded with DAF-2DA with and without L-NAME, an NOS inhibitor. Upon probe loading, the ester bonds of DAF-2DA are hydrolyzed by intracellular esterases, generating DAF-2, which accumulates within the cell. DAF-2 is nonfluorescent until oxidized by NO. As described in the calibration process (Supporting Information, p S-3), the detection limits of fluorescence-based NO measurement within the ModCR were 2 and 10 nM for cell-free solutions and intracellular NO, respectively. Using the calibration curve (Supporting Information Figure S-1b), the population-averaged FI signals (n = 600 cells) were estimated to be equivalent to NO concentrations of 100 and 200 nM in nondifferentiated and differentiated cell populations, respectively. In the presence of NOS inhibitor, an average decrease of about 25% in mean FI values was evident in both cell populations, which indicates that only one-quarter of the FI signal originates from de novo enzymatic production of NO. Taken together, these data indicate that, under experimental conditions used here, the enzyme-dependent NO level in nondifferentiated U937 cells ranges in the tens of nanomoles per liter (10−30 nM), whereas in PMA-differentiated cells NOS-based concentration is about doubled (30−50 nM). 7317

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Figure 2. Overlapping images of fluorescence and bright-field images of representative individual nondifferentiated (left panel) and differentiated (right panel) U937 cells within the picowell array. Cells were simultaneously stained with DAF-2 (a) and ThiolTracker Violet (b). Representative scatter diagrams of DAF-2 FI vs ThiolTracker Violet FI measured in nondifferentiated (top) and differentiated (bottom) individual U937 cells. Red symbols represent population-averaged values (c). Distribution histograms of NO ratio (ratio between intracellular NO level after and before activation) of differentiated U937 cell populations either activated by PMA, LPS, or nonactivated (d).

However, given the well-reported difficulties in the absolute measurement of NO concentration, and the fact that NO modulates on its own synthesis, these fluorescence measurements can only provide information on relative rates and dynamics. Overlapping images of fluorescent and bright-field images of nondifferentiated (Figure 2a, left) and differentiated (Figure 2a, right) U937 cells loaded with DAF-2DA and settled at the bottom of the array are presented. Differentiated cells exhibited morphological alteration (Figure 2a, right), and their population-averaged DAF-2 fluorescent signal was significantly higher than that of the nondifferentiated cell group (100.2 ± 52.2 and 83.1 ± 24.8 au for differentiated and nondifferentiated cells, respectively, p < 0.005, n = 1200). Variation in individual FI values within a cell population was higher in differentiated cells. Homogeneity of cellular staining as reflected by the variance of all pixel values in a single-cell area was the same in both groups (p = 0.22). Since cellular heterogeneity of NO levels may be related to the intracellular redox state, the concentration of GSH was measured concomitantly with intracellular NO using ThiolTracker Violet (Figure 2b). Correlation between basal levels of GSH and NO was found neither in nondifferentiated nor in differentiated cells (Figure 2c) at the single-cell resolution (Pearson correlation coefficients 0.48 and 0.28 for nondifferentiated and differentiated cells, respectively). However, averaged FI signal of GSH probe (n =

400) was significantly lower following cell differentiation (8.85 ± 3.3 au in nondifferentiated and 7.42 ± 4 au in differentiated cells, p < 0.001). Measurement of NO Levels Following On-Array Cell Activation. Activated human macrophages have been shown to respond to external stimuli such as bacterial LPS and γinterferon and to produce NO which promotes cytotoxic action.16,19 Human monocytes can also be stimulated by PMA, by activating protein kinase C which is involved in induction of NO production and iNOS triggering pathways.20−22 Differentiated U937 cells preloaded with DAF-2DA were introduced to the ModCR and then activated in the presence of PMA and LPS for 30 min. The unique picowell structure allows measurement of FI before and after activation, thus enabling calculation of relative change in NO content for each individual cell. The ratio between intracellular NO level after and before activation was termed NO ratio. As seen in the distribution histograms (Figure 2d), following cell activation, distribution curves of NO ratio shifted to higher values. Population-averaged NO ratio of PMA-activated cell populations was significantly higher than the average ratio measured in nonactivated cells (1.11 ± 0.20 and 0.95 ± 0.14 au with and without activation, respectively, p < 0.0005). Upon LPS activation, the rise in population-averaged NO ratio values was more pronounced, indicating a major increase in NO concentration (mean relative ratio 1.60 ± 0.91 au) (Figure 2d). The NO ratio parameter normalizes initial 7318

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Figure 3. (a) Time-dependent FI values measured in seven representative individual cells upon PMA activation. Each dot represents the FI of one individual cell at a given time point. (b) Normalized time response curves of FI of differentiated (red) and nondifferentiated (black) cell populations upon PMA activation. Each dot represents an averaged normalized FI ± SD of 500 cells. Arrow indicates time of stimulant addition. White squares and black triangles represent addition of DMSO and PMA plus L-NAME, respectively. (c) Distribution histograms of NO ratio values within PMAactivated and nonactivated U937 cell populations. (d) Time response of NO level changes measured in HR and LR subgroups of U937 cells upon PMA activation (left panel) and DMSO addition (right panel). Arrows indicate time of stimulant addition. Numbers indicate percent of cell group out of total cell number. Note the absence of the HR group in DMSO-treated cells. Asterisks indicate statistically significant difference between the two subgroups.

inherent variability between individual cells which arise from changes in intracellular pH gradients, different dye uptake, or de-esterification of DAF-2DA by various cells. It was shown that esterase activity, which can vary even between normal and drug-resistant cells in the same cell line,28 can potentially influence probe loading and leakage of oxidized probe. These limitations are overcome by using the picowell technology, which facilitates monitoring each cell before, during, and after treatment and relates the results at each time point to the initial basal value. Hence, the difference in measured cellular FI is independent of dye uptake or esterase reaction rates of individual cells. Short-Term Kinetics of NO Generation Rates. For short-term kinetic analysis, U937 cells preloaded with DAF2DA were introduced into the picowell array and the first image was acquired. Then, stimulant or its solvent (DMSO, PBS) was added, and a series of images were taken at 30 s intervals during a 3 min period. FI(t) curves of seven representative individual nondifferentiated U937cells, all simultaneously measured every 30 s, are shown in Figure 3a. The individual curves differ in appearance

and maximum values, indicating cellular heterogeneity both in NO generation rate and NO level. Normalizing individual cell FI to baseline values at each time point (t = 0) revealed the kinetics of NO generation upon activation. Normalized population-averaged FI(t) response curves of NO production in nondifferentiated and differentiated U937 cell groups treated either with DMSO, PMA alone, or PMA and L-NAME are shown in Figure 3b. DMSO alone did not induce any alterations in mean FI. Differentiated promonocytes showed an increase in averaged FI immediately following addition of PMA. A minimal increase in population-averaged intracellular FI signal was detected at the first measurement following exposure of nondifferentiated U937 cells to PMA, which decays thereafter as a result of fading. Introduction of L-NAME concomitantly with PMA abolished the PMA-induced increase in FI and showed drastic FI decay upon repeated illumination of cells. The density histogram of NO ratio values measured in nondifferentiated promonocytes is shown in Figure 3c. In the nonactivated cell population, mean NO ratio was 0.958 ± 0.07 au, indicating that no change had occurred in the NO 7319

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formation rate. However, in activated cells, a subgroup of cells was revealed which showed higher relative rates (Figure 3c). When choosing an NO ratio of 1.03 as a cutoff level (mean + SD of the control cell population), about 40% of the activated U937 cells exhibited a measurable increased NO production rate, and were termed “high-reacting group” (HR), whereas cells showing no change or a decrease in intracellular FI upon activation were termed “low-reacting cell group” (LR) (Figure 3c). The HR cell group was evident only upon cell activation and comprised 20−60% of total cell number. Conversely, under the same experimental conditions, following cell treatment either with DMSO or with PBS the HR subpopulation was either absent or never exceeded 10% of total cell number (Figure 3d). Monitoring the NO production rate for up to 30 min revealed that the LR group did not produce an equivalent amount of NO during this time period (Supporting Information). Mean FI(t) measured in the HR cluster was significantly higher than the mean FI(t) of the LR group (Figure 3d) (1.23 ± 0.16 au for the HR group and 0.925 ± 0.079 au for the LR group, p < 0.005). However, the two groups did not differ significantly in their cell size (mean cell area 990.8 ± 118 and 1080.2 ± 151 au in low- and high-reactive subpopulations, respectively, p > 0.05). GSH level measured concomitantly in the same cells was significantly lower in the HR group (8.22 ± 2.2 au for the HR group and 9.54 ± 2.2 au for the LR group, p = 0.004), indicating that cellular heterogeneity in NO generation may be partially related to intracellular reduced glutathione levels. These results are in agreement with previous data which showed that GSH can regulate protein complex nuclear factor κ B (NFκB) activation. Thus, low level of GSH activates the NFκB pathway and leads to up-regulation of iNOS and other survival genes.37,38 NOS Expression Levels upon Cell Activation. In order to estimate protein amount, U937 cells were first activated within the ModCR, and NO levels were measured. Then, IHC staining of iNOS enzyme in the same individual cells was done and protein level was determined. Individual promonocyte cells retained their spatial location during cell fixation, permeabilization, and immunostaining (Figure 4). There is high heterogeneity in enzyme quantity, in both nondifferentiated and differentiated cells, suggesting differences in expression levels of iNOS (CV within cell population about 100%). Figure 5 depicts the density histogram of individual cell iNOS FI values in nondifferentiated cells without, and following, PMA activation. Upon activation, up-regulation of iNOS was evident, and a cell group having an increased amount of iNOS was discovered. Population-averaged FI values were 35.8 ± 39.5 and 28.8 ± 28.41 au for activated and nonactivated cells, respectively, p > 0.05. High standard deviations were previously observed for single cells at the cDNA,35 mRNA,36 and protein levels.34,35 Recently, it has become clear that such cellular heterogeneity within cell population is a widespread event which substantially arises from stochastic expression of genes and proteins.34 Analytical tools, like the ModCR, which enable direct measurement in single cells, are essential for understanding these complex processes. The unique ModCR facilitates correlation between NO production rates in each individual cell with its corresponding iNOS level. Baseline protein quantity per cell was not significantly correlative or predictive for either the basal or induced NO rate of production in nondifferentiated or differentiated cell populations. Nevertheless, upon cell

Figure 4. (a) Transmitted light and (b) fluorescence micrographs of individual U937 cells loaded with DAF-2DA and (c) expression of iNOS in the same individual cells observed following fixation, permeabilization, and IHC staining.

activation, the HR subgroup exhibited enzyme regulation different from that of the LR subgroups. Distribution of individual cell anti-iNOS FI values measured in subgroups of U937 cells having high and low NO rate of generation (HR and LR) is presented (Figure 5, parts c and d). As seen, increased iNOS levels in PMA-activated cells can be attributed mainly to the HR cell group, since their mean enzyme level was significantly higher than that of the LR subpopulation (49.7 ± 35.1 and 30.7 ± 29.3 au in HR and LR, respectively, p = 0.01), the latter group showing an iNOS expression level similar to that of nonactivated cells. Heterogeneity in NO production and its following modulation by human monocytes have been previously described.29,30 However, correlation between spontaneous or induced NO production and other cellular parameters was never tested at individual cell resolution. Such analyses reveal that the association between enzyme expression and their product levels depends on specific conditions of cell and stimulant. The fact that no significant correlation was found at the single-cell resolution between protein quantity, NO production rates, and GSH levels indicates that additional factors are involved in NO intracellular concentration, some of which may be the other isoenzymes and NO formation from inorganic anions.13 Modulation of Enzyme Expression by Exogenous NO and LPC. For validation of the current methodology, the effects of exogenous NO and LPC on synthesis of intracellular NO were tested. LPC is an atherogenic phospholipid generated during LDL oxidation23 and has been shown to modulate NO production by up-regulating eNOS in endothelial cells.24,25 In order to examine the regulation NO levels on its synthesis, probe-loaded cells were introduced into the ModCR and 7320

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a concentration-dependent outcome of NO on its own synthesis.39 Conversely, following exposure of nondifferentiated U937 cells to LPC, only minor elevation in averaged NO ratio was evident (1.005 ± 0.99 and 0.976 ± 0.91 au for LPC-treated and nontreated cells, respectively, p = 0.02). However, a significant up-regulation of iNOS was presented by the corresponding IHC analysis of the same cells. FI values of anti-iNOS in LPC-treated cells increased 2-fold compared to nontreated cells (26.3 ± 2.27 and 12.3 ± 1.10 au for LPCtreated and nontreated cells, respectively, p < 0.0005). LPCinduced up-regulation of iNOS was evident also when U937 cells were exposed to LPC in the presence of L-NAME (25.2 ± 20.1 au), suggesting a modulating mechanism not dependent on local NO concentration. Apparently, such effects of LPC on NO generation in promonocyte cells have not been previously demonstrated.

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CONCLUSIONS Advanced technological and analytical capabilities are introduced for fluorescence and bright-field investigation of individual live cells, followed by IHC study of the same cells. For NO-producing cells, such examination reveals the relation between NOS levels, their activity, and antioxidant concentrations in each cell within the population. In addition, detection of fast kinetics and low intracellular NO signals is facilitated, enabling identification of specific cells, or subgroups of cells, seemingly important for the study of NO mechanisms, as well as for RNS-related drugs and treatment. Other potential applications of this technology may involve research fields which utilize numerous unanchored nonadherent cell types (immunology, hematology, autoimmunity, transplant rejection) as well as ex vivo analysis of intact liquid tissues and primary individual cells derived from bodily fluids (peripheral blood, bone marrow, CSF, synovial fluid, urine). In addition, multisized nonadherent biological cells (including prokaryotic cells like bacteria and yeast) can be investigated. Finally, current technology facilitates cellular communication either via secreted molecules or by direct cell−cell contact. Individual cells within the picowells share common space, and distance communication by active molecules (chemokines, cytokines) is enabled. By adjusting the dimensions of picowells to hold cell doublets, small groups of cells, or multicellular structures (spheroids), direct cellular interactions are possible and can be monitored at a single-cell resolution.18,40

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Figure 5. Distribution histograms of iNOS2 levels in individual U937 cells (a) before and (b) following activation. (c) LR and (d) HR distribution histograms of iNOS levels in subpopulations of the nondifferentiated U937 cells upon PMA activation.

ASSOCIATED CONTENT

S Supporting Information *

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

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exposed to various concentrations of NO donor. Time response of changes in NO levels measured in U937 cell populations upon exposure to DETA/NO is presented in the Supporting Information (Figure S-2a). IHC analysis of enzyme expression in the same cells indicated that 60 min of exposure to NO donor modulated expression of iNOS in a dose-dependent manner. Low NO concentrations induced insignificant increase in protein levels, while exposure of nondifferentiated U937 cells to 1 μM NO resulted in significant enhancement of enzyme expression (mean FI 38.4 ± 14.6 and 46.7 ± 13.5 au, in nontreated cells exposed to 1 μM NO, respectively, p < 0.0005). Results agree with previous data, which demonstrated

AUTHOR INFORMATION

Corresponding Author

*Phone: 972-3-534-4675. Fax: 972-3-534-2019. E-mail: motti. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study has been made possible through the bequest of Moshe-Shimon and Judith Weisbrodt. 7321

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dx.doi.org/10.1021/ac202741z | Anal. Chem. 2012, 84, 7315−7322