Carbon-fiber Microelectrode Amperometry Research Articles

Platelet Response to Allergens, CXCL10, and CXCL5 in the Context of Asthma.

Asthma is a chronic respiratory disease initiated by a variety of factors, including allergens. During an asthma attack, the secretion of C-X-C-motif chemokine 10 (CXCL10) and chemokine ligand 5 (CCL5) causes the migration of immune cells, including platelets, into the lungs and airway. Platelets, which contain three classes of chemical messenger-filled granules, can secrete vasodilators (adenosine diphosphate and adenosine triphosphate), serotonin (a vasoconstrictor and a vasodilator, depending on the biological system), platelet-activating factor, N-formylmethionyl-leucyl-phenylalanine ((fMLP), a bacterial tripeptide that stimulates chemotaxis), and chemokines (CCL5, platelet factor 4 (PF4), and C-X-C-motif chemokine 12 (CXCL12)), amplifying the asthma response. The goal of this work was threefold: (1) to understand if and how the antibody immunoglobulin E (IgE), responsible for allergic reactions, affects platelet response to the common platelet activator thrombin; (2) to understand how allergen stimulation compares to thrombin stimulation; and (3) to monitor platelet response to fMLP and the chemokines CXCL10 and CCL5. Herein, high-pressure liquid chromatography with electrochemical detection and/or carbon-fiber microelectrode amperometry measured granular secretion events from platelets with and without IgE in the presence of the allergen 2,4,6-trinitrophenyl-conjugated ovalbumin (TNP-Ova), thrombin, CXCL10, or CCL5. Platelet adhesion and chemotaxis were measured using a microfluidic platform in the presence of CXCL10, CCL5, or TNP-OVA. Results indicate that IgE binding promotes δ-granule secretion in response to platelet stimulation by thrombin in bulk. Single-cell results on platelets with exogenous IgE exposure showed significant changes in the post-membrane-granule fusion behavior during chemical messenger delivery events after thrombin stimulation. In addition, TNP-Ova allergen stimulation of IgE-exposed platelets secreted serotonin to the same extent as thrombin platelet stimulation. Enhanced adhesion to endothelial cells was demonstrated by TNP-Ova stimulation. Finally, only after incubation with IgE did platelets secrete chemical messengers in response to stimulation with fMLP, CXCL10, and CCL5.

Plasmodium chabaudi Affects Mast Cell Degranulation as Measured by Carbon-Fiber Microelectrode Amperometry.

Mast cells (MCs) are effector cells of the immune system commonly known for their role in asthma and allergy. They are present throughout biological systems in various tissues, serving as an interface between the biological system and environment. Previous work characterizing the impact of malaria on MCs revealed contradictory results, showing minimal to strong correlation between MC degranulation and disease progression. This work seeks to reveal how MC degranulation is impacted in the presence of malaria, induced by Plasmodium chabaudi, using a mouse model and a single cell measurement technique that reveals exquisite biophysical detail about any impacts to the degranulation process. It was hypothesized that the malaria parasites would impact MC degranulation response during live infection, and the differences would be revealed via carbon-fiber microelectrode amperometry. In fact, the data collected show that different stages of malaria infection affect MC degranulation differently, affirming the importance of considering different infection stages in future studies of malarial immune response. Furthermore, a comparison of MC degranulation response to that measured from platelets under similar circumstances shows similar trends in quantitative degranulation, suggesting that MC and platelet exocytosis machinery are affected similarly despite their distinct biological roles. However, based on the small number of mouse replicates, the studies herein suggest that there should be further study about cellular and disease processes. Overall, the work herein reveals important details about the role of MCs in malaria progression, relevant during treatment decisions, as well as a potentially generalizable impact on chemical messenger secretion from cells during malarial progression.

Antimalarial drugs impact chemical messenger secretion by blood platelets

BackgroundAdvances in antimalarial drug development are important for combating malaria. Among the currently identified antimalarial drugs, it is suggested that some interact directly with the malarial parasites while others interact indirectly with the parasites. While this approach leads to parasite elimination, little is known about how these antimalarial drugs impact immune cells that are also critical in malarial response. MethodsHerein, the effects of two common antimalarial drugs, chloroquine and quinine, on platelets were explored at both the bulk level, using high performance liquid chromatography, and the single cell level, using carbon-fiber microelectrode amperometry, to characterize any changes in chemical messenger secretion. ResultsThe data reveal that both drugs cause platelet activation and reduce the number of platelet exocytosis events as well as delay fusion pore opening and closing. ConclusionsThis work demonstrates how chloroquine and quinine quantitatively and qualitatively impact in vitro platelet function. General significanceOverall, the goal of this work is to promote understanding about how antimalarial drugs impact platelets as this may affect antimalarial drug development as well as therapeutic approaches to treat malarial infection.

Insight into the Effects of Plasmodium chabaudi on Platelets Using Carbon-Fiber Microelectrode Amperometry.

Platelets are anuclear circulating cell bodies within the bloodstream commonly known for their roles in clot formation during vascular injury to prevent blood loss. They also have significant impact in a range of diseases, including malaria. However, the role of platelets in malaria is controversial, with contradicting evidence suggesting either that they assist in destruction of malarial parasites or facilitate a severe form of malaria. Precedent work suggests that the timing of infection is critical in determining whether platelets switch roles from being protective to deleterious. As such, the work herein makes use of the unique mechanistic perspective offered by carbon-fiber microelectrode amperometry (CFMA) to understand how platelet secretion is impacted in malarial infection stages (ascending parasite count versus descending parasite count). Malarial platelet behavior was compared to platelets from noninfected control mice by probing their exocytotic function. Results suggest that mouse malaria caused by the parasite Plasmodium chabaudi, during both ascending and descending infection stages, reduces platelet exocytotic events and delays platelet granule fusion; in addition, platelets are more impacted by the disease early in the infection stages. In all, understanding platelet behavior in the malarial context may present new therapeutic routes to treat or cure malaria.

Variations in Fusion Pore Formation in Cholesterol-Treated Platelets

Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 μs time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.

Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

Analytical characterization of the role of phospholipids in platelet adhesion and secretion.

The cellular phospholipid membrane plays an important role in cell function and cell–cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography–tandem electrospray ionization mass spectrometry (UPLC–MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC–MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

TiO2 nanoparticle-induced ROS correlates with modulated immune cell function

Design of non-toxic nanoparticles will be greatly facilitated by understanding the nanoparticle-cell interaction mechanism on a cell function level. Mast cells are important cells for the immune system’s first line of defense, and we can utilize their exocytotic behavior as a model cellular function as it is a conserved process across cell types and species. Perturbations in exocytosis can also have implications for whole organism health. One proposed mode of toxicity is nanoparticle-induced reactive oxygen species (ROS), particularly for titanium dioxide (TiO2) nanoparticles. Herein, we have correlated changes in ROS with the perturbation of the critical cell function of exocytosis, using UV light to induce greater levels of ROS in TiO2 exposed cells. The primary culture mouse peritoneal mast cells (MPMCs) were exposed to varying concentrations of TiO2 nanoparticles for 24 h. ROS content was determined using 2,7-dihydrodichlorofluorescein diacetate (DCFDA). Cellular viability was determined with the MTT and Trypan blue assays, and exocytosis was measured by the analytical electrochemistry technique of carbon-fiber microelectrode amperometry. MPMCs exposed to TiO2 nanoparticles experienced a dose-dependent increase in total ROS content. While there was minimal impact of ROS on cellular viability, there is a correlation between ROS amount and exocytosis perturbation. As nanoparticle-induced ROS increases, there is a significant decrease (45 %) in the number of serotonin molecules being released during exocytosis, increase (26 %) in the amount of time for each exocytotic granule to release, and decrease (28 %) in the efficiency of granule trafficking and docking. This is the first evidence that nanoparticle-induced ROS correlates with chemical messenger molecule secretion, possibly making a critical connection between functional impairment and mechanisms contributing to that impairment.

Exploring airway smooth muscle-mediated mast cell function at the single cell level using carbon-fiber microelectrode amperometry (177.6)

Abstract An important distinguishing feature of asthma pathology is the migration of mast cells (MCs) into the airway smooth muscle (ASM). MCs are known to influence the immune response in a variety of capacities, including the release of many stored immunoactive mediators from secretory vesicles via exocytosis. Conversely, ASM is known to produce several mediators that potentially influence MC activity including the chemoattractant CXCL10. However, the specific consequences of interaction between MCs and ASM on asthma pathophysiology have yet to be fully explored. Carbon-fiber microelectrode amperometry (CFMA) is a unique analytical tool that allows the direct and label free detection of secreted serotonin as it is released from a single MC. Individual packets of serotonin corresponding to single vesicle fusion events are manifested as discrete current spikes in the collected amperometric trace. Individual spike analysis of each trace provides information such as vesicular content and delivery capacity, vesicle fusion efficiency and other kinetic information pertaining to exocytosis regulation. To explore the relationship between MCs and ASM in asthma, CFMA was used to monitor the effects of ASM and asthma-related factors, such as the cytokines CXCL10 and RANTES, on MC function in vitro. Analysis of CFMA data has shown significant modulation of MC secretory function in response to these factors, demonstrating the value of CFMA for the study of MC-related inflammatory processes.

Carbon-Fiber Microelectrode Amperometry Reveals Sickle-Cell-Induced Inflammation and Chronic Morphine Effects on Single Mast Cells

Sickle cell disease, caused by a mutation of hemoglobin, is characterized by a complex pathophysiology including an important inflammatory component. Mast cells are tissue-resident leukocytes known to influence a range of immune functions in a variety of different ways, largely through the secretion of biologically active mediators from preformed granules. However, it is not understood how mast cells influence the inflammatory environment in sickle cell disease. A notable consequence of sickle cell disease is severe pain. Therefore, morphine is often used to treat this disease. Because mast cells express opioid receptors, it is pertinent to understand how chronic morphine exposure influences mast cell function and inflammation in sickle cell disease. Herein, carbon-fiber microelectrode amperometry (CFMA) was used to monitor the secretion of immunoactive mediators from single mast cells. CFMA enabled the detection and quantification of discrete exocytotic events from single mast cells. Mast cells from two transgenic mouse models expressing human sickle hemoglobin (hBERK1 and BERK) and a control mouse expressing normal human hemoglobin (HbA-BERK) were monitored using CFMA to explore the impact of sickle-cell-induced inflammation and chronic morphine exposure on mast cell function. This work, utilizing the unique mechanistic perspective provided by CFMA, describes how mast cell function is significantly altered in hBERK1 and BERK mice, including decreased serotonin released compared to HbA-BERK controls. Furthermore, morphine was shown to significantly increase the serotonin released from HbA-BERK mast cells and demonstrated the capacity to reverse the observed sickle-cell-induced changes in mast cell function.

Cholesterol effects on vesicle pools in chromaffin cells revealed by carbon-fiber microelectrode amperometry

Cell-cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.

Effect of polymer deposition method on thermoresponsive polymer films and resulting cellular behavior.

Poly(N-isopropyl acrylamide) or pNIPAM is a thermoresponsive polymer that is widely studied for use in bioengineering applications. The interest in this polymer lies in the polymer's unique capability to undergo a sharp property change near physiological temperature, which aids in the spontaneous release of biological cells from substrates. Currently, there are many methods for depositing pNIPAM onto substrates, including atom-transfer radical polymerization (ATRP) and electron beam ionization. Each method yields pNIPAM-coated substrates with different surface characteristics that can influence cell behavior. In this work, we compare two methods of pNIPAM deposition: plasma deposition and codeposition with a sol-gel. The resulting pNIPAM films were analyzed for use as substrates for mammalian cell culture based on surface characterization (XPS, ToF-SIMS, AFM, contact angles), cell attachment/detachment studies, and an analysis of exocytosis function using carbon-fiber microelectrode amperometry (CFMA). We find that although both methods are useful for the deposition of functional pNIPAM films, plasma deposition is much preferred for cell-sheet engineering applications because of the films' thermoresponse, minimal change in cell density, and maintenance of supported cell exocytosis function.

Understanding the Fundamentals of Platelet Granular Storage and Release at Single Cell Level

Platelets are traditionally recognized as critical cells in hemostasis and thrombosis but have also recently been identified to play a significant role in many diseases, including bacterial infections, cancer, and allergic asthma. Platelets store and release important messenger molecules via exocytosis from two populations of granules (α- granules with adhesive protein species and δ- granules with small molecule/ion species). However, very little known about how chemical messengers are stored in these granules and the driving forces for secretion into the blood stream, thus limiting the development of new therapeutic approaches to manage the role of platelets in many physiological events. Carbon-fiber microelectrode amperometry (CFMA) enables both quantal and kinetic analysis of the exocytotic event of single cell with sub-ms time resolution but is limited to measuring electroactive species from δ-granules. Localized surface plasmon resonance (LSPR) spectroscopy is a complementary tool that offers an outstanding sensing ability by measuring the extremely small changes in the sensing media. This work aims to overcome the gap in fundamental knowledge about platelet granular storage and secretion by detecting serotonin secretion from δ- granules using CFMA and Platelet factor 4 (PF4) secretion from α- granules using LSPR. These measurements are performed while exposing platelets to different stimulants, including ionomycin, adenosine diphosphate (ADP), and thrombin. Significant differences are seen in both α- and δ-granule secretion with varied stimulant exposure, and these combined measurements yield biophysical insight into the mechanism behind these secretory behaviors.

Mechanistic Insights into Exocytosis Dysfunction After Noble Metal Nanoparticle Exposure

As nanoparticle use in consumer products and medical applications continues to increase drastically, the field of nanotoxicology has developed to examine the impacts of these nanoparticles in biological systems. Previous work,[1, 2] with primary culture chromaffin and mast cells exposed to Au and Ag nanoparticles, has shown a general trend for nanoparticle effects on cellular exocytosis. That work showed that 24 hours of exposure, to nanoparticles at concentrations from 0.1 to 10 nM for nanoparticles, results in fewer molecules being secreted over a longer period of time. This work investigates the underlying causes of these changes in exocytosis in nanoparticle-exposed cells. Several possible mechanisms of dysfunction could be related to these changes, including altered membrane properties, reactive oxygen species (ROS) generation, or changes in calcium signaling. To examine these potential mechanisms of exocytosis dysfunction, carbon-fiber microelectrode amperometry (membrane properties) and fluorescence methods (vesicle matrix-nanoparticle interactions, calcium signaling, and ROS generation) were employed. Preliminary results indicate there were no significant changes in calcium signaling following nanoparticle exposure, nor were there changes in the pre-spike foot occurrence, which would indicate an alteration in the membrane properties post-exposure. However, preliminary results suggest that there were changes in the rate of ROS generation, which could impair exocytosis machinery in addition to having other downstream effects. 1. Love, S. A.; Haynes, C. L., Assessment of functional changes in nanoparticle-exposed neuroendocrine cells with amperometry: exploring the generalizability of nanoparticle-vesicle matrix interactions. Analytical and Bioanalytical Chemistry 2010, 398 (2), 677-88. 2. Marquis, B.; Maurer-Jones, M.; Braun, K.; Haynes, C., Amperometric assessment of functional changes in nanoparticle-exposed immune cells: varying Au nanoparticle exposure time and concentration. Analyst 2009, 134 (11), 2293-300.

Investigation of noble metal nanoparticleζ-potential effects on single-cell exocytosis function in vitro with carbon-fiber microelectrode amperometry

Since noble metal nanoparticles are increasingly found in consumer goods, there is a need for information about potential impacts of these nanoparticles on cellular function to avoid environmental and health risks associated with exposure. In this study, spherical Au and Ag nanoparticles of similar size were synthesized and modified to assess the effects of ζ-potential on immune cell function. Nanoparticle ζ-potential was controlled by employing surfactant exchange to generate nanoparticles with positive or negative surface charge. Mouse peritoneal mast cells (MPMCs) were then exposed to 5-15 µg ml(-1) of these nanomaterials, and uptake was assessed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Uptake for positively charged nanoparticles was more efficient than for negatively charged nanomaterials, and all nanoparticles were taken up in a concentration-dependent manner. Following uptake, MPMC degranulation function was assessed using carbon-fiber microelectrode amperometry (CFMA), showing decreased quantal secretion of serotonin by MPMCs exposed to the positively charged Au nanoparticles and negatively charged Ag nanoparticles. The overall efficiency of the degranulation process (indicated by amperometric spike frequency) decreased for all Au-exposed MPMCs. However, only the negatively charged version of the Ag nanomaterial resulted in decreased MPMC degranulation efficiency. Further studies revealed that ionic Ag was partially responsible for the observed effects. Overall, these studies reveal the complex nature of interactions between noble metal nanomaterials and cells that result in perturbed cellular function and illustrate the necessity of thorough nanoparticle characterization for interpretation of cellular function assays.

Evaluating the effects of immunotoxicants using carbon fiber microelectrode amperometry

Carbon fiber microelectrode amperometry (CFMA) is explored as a technique for studying the effects of immunotoxicants on single-cell in vitro exocytosis function in a mouse peritoneal mast cell (MPMC)/fibroblast co-culture model. MPMCs were acutely exposed to between 10 and 100μM of the immunotoxicants mono-2-ethylhexyl phthalate (MEHP) and bisphenol A (BPA), and release of serotonin was evaluated by CFMA. A significant decrease in the quantal content of serotonin was measured for all levels of exposure to both MEHP and BPA. The overall efficiency of the exocytotic function of MPMCs was found to be impaired by all exposure concentrations of BPA, but this efficiency was only impaired at the lowest exposure concentration of MEHP. This study illustrates the potential of CFMA as a technique for determining quantitative and biophysical chemical information in in vitro immunotoxicological studies.

Functional Assessment of Metal Oxide Nanoparticle Toxicity in Immune Cells

Understanding the nanoparticle-cell interaction is critical for the safe development of nanomaterials. Herein, we explore the impact of three metal oxide nanoparticles, nonporous Stober SiO(2), mesoporous SiO(2), and nonporous anatase TiO(2) nanoparticles, on primary culture mast cells. Using transmission electron microscopy and inductively coupled plasma atomic emission spectroscopy, we demonstrate that each class of nanoparticle is internalized by the mast cells, localizing primarily in the secretory granules, with uptake efficiency increasing in the following order: nonporous SiO(2) < porous SiO(2) < nonporous TiO(2) nanoparticles. The influence of nanoparticle-laden granules was assessed using carbon-fiber microelectrode amperometry measurements that reveal functional changes in chemical messenger secretion from mast cell granules. Both nonporous and porous SiO(2) nanoparticles cause a decrease in the number of molecules released per granule, with nonporous SiO(2) also inducing a decrease in the amperometric spike frequency and, therefore, having a larger impact on cell function. As the two classes of SiO(2) nanoparticles vary only in their porosity, these results suggest that, while the mesoporous SiO(2) has a drastically larger total surface area due to the pores, the cell-contactable surface area, which is higher for the nonporous SiO(2), is more important in determining a nanoparticles' cellular impact. In comparison, exposure to nonporous TiO(2) slows the kinetics of secretion without altering the number of molecules released from the average granule. The varying immune cell response following exposure to nonporous SiO(2) and nonporous TiO(2) indicates that the nanoparticle-cell interactions are also modulated by surface chemistry.

Assessment of functional changes in nanoparticle-exposed neuroendocrine cells with amperometry: exploring the generalizability of nanoparticle-vesicle matrix interactions

Using two of the most commonly synthesized noble metal nanoparticle preparations, citrate-reduced Au and Ag, the impacts of short-term accidental nanoparticle exposure are examined in primary culture murine adrenal medullary chromaffin cells. Transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Alamar Blue viability studies revealed that nanoparticles are taken up by cells but do not decrease cell viability within 48 hours of exposure. Carbon-fiber microelectrode amperometry (CFMA) examination of exocytosis in nanoparticle-exposed cells revealed that nanoparticle exposure does lead to decreased secretion of chemical messenger molecules, of up to 32.5% at 48 hours of Au exposure. The kinetics of intravesicular species liberation also slows after nanoparticle exposure, between 30 and 50% for Au and Ag, respectively. Repeated stimulation of exocytosis demonstrated that these effects persisted during subsequent stimulations, meaning that nanoparticles do not interfere directly with the vesicle recycling machinery but also that cellular function is unable to recover following vesicle content expulsion. By comparing these trends with parallel studies done using mast cells, it is clear that similar exocytosis perturbations occur across cell types following noble metal nanoparticle exposure, supporting a generalizable effect of nanoparticle-vesicle interactions.

Real time, single-cell characterization of basophilic exocytosis through carbon-fiber microelectrode amperometry (CFMA) (42.1)

Abstract Histamine released by previously sensitized basophils plays a crucial role in inflammation and immediate hypersensitivity reactions. Whereas ensemble-based immunohistochemical assays characterize the average release profile of cell populations, differences among individual basophils or basophil sub-populations remain largely unexplored based on technological limitations. Previous work in our lab has used microelectrochemical techniques to measure real time quantal release from single cells isolated from separate platelet and mast cell populations. Herein we report the use of carbon-fiber microelectrodes to measure the concentration and time course of single-cell histamine secretion by primary culture basophils in response to varying physiological and electrical stimuli. Future work will utilize CFMA and the patch clamp technique to probe single-cell degranulation in a polydimethylsiloxane (PDMS) microfluidic chip model of graduated complexity.

The effects of co-culture of fibroblasts on mast cell exocytotic release characteristics as evaluated by carbon-fiber microelectrode amperometry

In this work, carbon-fiber microelectrode amperometry (CFMA) is employed to probe changes in the biophysical mechanism of exocytosis under varied cell culture conditions. Degranulation and serotonin exocytosis from mouse peritoneal mast cells (MPMCs) were measured both without and with co-cultured Swiss-albino 3t3 fibroblasts using CFMA. After 24 h in culture, there are distinct differences in the exocytotic characteristics of MPMCs cultured with and without fibroblast support cells, as detected by CFMA, including an increased number of secreted serotonin molecules, number of granule fusion events, secretion rate, and granule membrane tension. Beyond 48 h in culture, MPMCs cultured alone cannot be analyzed using CFMA due to decreased viability and membrane tension whereas MPMCs co-cultured with fibroblasts were maintained for up to 28 days in culture. Some secretion characteristics evolved over the long-term co-culture but the total amount of serotonin released per cell remained largely constant. This work quantitatively demonstrates that the MPMC/fibroblast co-culture system presents a promising model system for chronic exposure or disease model studies based on CFMA analysis.