Single Mast Cells Research Articles

Гистологические особенности ряда органов куньих и их патологические изменения при неэкссудативном перитоните

The article presents the histological features of some organs of weasel family. Also described the pathological changes during nonexudative form of peritonitis of weasel family. The studies were carried out at the Department of General Pathology named after V. M. Koropov of Moscow State Academy of Veterinary Medicine and Biotechnology – MBA named after K. I. Scriabin. The objects of investigation were predatory animals weasel family both healthy and with sings of peritonitis. There are 21 sables, 11 mink cellular content and 15 domestic ferrets. In the study complex methodological approach was used, including classical anatomical preparation, post mortem and histological examination. It has been established, that abdominal wall weasel has three puffs. There are muscle layer, a layer of dense connective tissue and serous membrane. The muscle layer is showing striated muscle tissue with endomysium layers. In the endomysia of muscle febers of abdominal wall of the weasel there are mast cell. Serous membrane is represented by mesothelium, which has typical structure of single – layered squamous epithelium. Large oil seal showing of fat cells with connective tissue layers, including lymphatic capillaries, vessels and nerve trunks. In a large oil seal single mast cell concentrated around the vessels and in the connective tissue of the omentum observed clusters of mast cells. Essential features of the structural organization of the stomach walls and intestinal canal are not found, but revealed a large number of goblet cells in the mucous membrane of the colon and small intestine and mycocytes in mucous membrane of stomach. The liver has a typical structure, lobilarity is weakly expressed. Liver beams are shortned, hepatocytes fit snugly together, sinusoid capillaries are broad. In morphological organization of organs of immune defense weasel significant features are not found. In the spleen was observed a lot of white pulp. In the cortical layer of lymph node was founded a large number of follicles. Thymus has sings age involution. Kidney and lungs weasel has not significant features. Predatory animals weasel family had predominantly nonexudative form of peritonitis. Under the nonexudative peritonitis form was observed sings of acute inflammation of many organs like as system inflammatory response syndrom.

Features of Remodeling of Prostate Stroma in Chronic Abacterial Prostatitis.

We performed a complex morphological study of biopsy samples of prostate gland from 30 men (mean age 46±11 years) with chronic abacterial prostatitis who were exposed unfavorable anthropogenic factors. The ultrastructural heterogeneity of smooth muscle cells was shown, among which cells with destructive changes in organelles predominated, solitary cells with signs of secretory function were revealed. Most endothelial cells in microvessels were characterized by low pinocytotic activity, destruction of organelles; focal hyperplastic ultrastructural changes were noted in some endotheliocytes. Along with fibroblasts, the stroma contained single mast cells; no inflammatory infiltration was revealed. The results of the study attested to complex mechanisms of remodeling of the prostate gland stroma in chronic abacterial prostatitis involving microvessels, smooth muscle cells, and probably mast cells, which determines the development of degenerative, destructive, and fibrotic processes.

Metastatic mast cell tumour in African hedgehog: A case report

Neoplastic lesions are common in African hedgehogs (Atelerix albiventris). To our knowledge, so far mast cell tumour metastases have only been described in local lymph nodes. An African hedgehog was referred to the veterinary clinic. A nodule was located on the right part of the mandible. Based on the clinical trial, an abscess was suspected. Surgery was performed, but a histopathological examination was not undertaken. After few months, tumour recurrence was detected and, additionally, a second tumour was found on the metatarsal skin. Due to the deteriorating clinical condition of the patient, euthanasia was decided, and a post-mortem examination was performed. During the post-mortem examination, a spleen tumour was detected, and, furthermore, mesenteric lymph nodes, liver and lung sections were also collected. The histopathological examination confirmed mast cell tumour metastasis to the spleen and lymph nodes, additionally single mast cells were found in the liver. Interestingly, the histological picture of the tumour does not show high malignancy. To our knowledge, this is the first description of a mast cell tumour with metastases to the spleen and lymph in the African hedgehog.

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis.

BackgroundSystemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells.MethodsFluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential.FindingsWe found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time.InterpretationThe KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

Click-Chemistry Based Allergen Arrays Generated by Polymer Pen Lithography for Mast Cell Activation Studies.

The profiling of allergic responses is a powerful tool in biomedical research and in judging therapeutic outcome in patients suffering from allergy. Novel insights into the signaling cascades and easier readouts can be achieved by shifting activation studies of bulk immune cells to the single cell level on patterned surfaces. The functionality of dinitrophenol (DNP) as a hapten in the induction of allergic reactions has allowed the activation process of single mast cells seeded on patterned surfaces to be studied following treatment with allergen specific Immunoglobulin E antibodies. Here, a click-chemistry approach is applied in combination with polymer pen lithography (PPL) to pattern DNP-azide on alkyne-terminated surfaces to generate arrays of allergen. The large area functionalization offered by PPL allows an easy incorporation of such arrays into microfluidic chips. In such a setup, easy handling of cell suspension, incubation process, and read-out by fluorescence microscopy will allow immune cell activation screening to be easily adapted for diagnostics and biomedical research.

Cell Surface-Anchored Fluorescent Probe Capable of Real-Time Imaging of Single Mast Cell Degranulation Based on Histamine-Induced Coordination Displacement.

Mast cells secrete histamine upon degranulation triggered by various stimuli. Herein, we report the new detection method of mast cell degranulation using the fluorescent probe capable of detection of the released histamine. The probe was designed as the Co(II) complex of a cyanine dye, which shows a turn-on fluorescence signal based on a histamine-induced coordination displacement mechanism. Fluorescence imaging using the cell surface-anchored fluorescent probe enabled the real-time detection of mast cell degranulation induced by various secretagogues.

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.

Mast Cells Positive to Tryptase Correlate with Angiogenesis in Early Breast Cancer.

Abstract Background: Literature data indicate that mast cells (MCs) are involved in tumor angiogenesis due to the release of several pro-angiogenetic factor among which tryptase, a serine protease stored in MCs granules, is one of the most active. In benign breast lesions and in primary breast cancer, tryptase-positive MCs are more numerous as compared to chymase-positive MCs. Recently, it has been demonstrated that tryptase-positive MCs are involved in angiogenesis in sentinel lymph nodes with micrometastases from patients with breast cancer. However no data has been published regarding the correlations between MCs positive to tryptase and angiogenesis in primary breast cancer tissue. In the present study, we have evaluated the correlations between the number of MCs positive to tryptase (MCDPT), the area occupied by MCs positive to tryptase (MCAPT), microvascular density (MVD) and endothelial area (EA) in a series of T1-3, N0-2, MO female breast cancer, by means of immunohistochemistry and image analysis methods.Material and Methods: Bioptic specimens were collected from 88 female breast cancer patients who had undergone breast cancer surgery. For the evaluation of MCDPT, MCAPT, MVD and EA, a three layer biotin-avidin-peroxidase system was utilized. Briefly, six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were obtained. Then sections were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with human-specific monoclonal antibodies anti-CD34 (QB-END 10; Bio-Optica Milan, Italy) diluted 1:50 for 1h at room temperature and anti-tryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1:100 for 1h at room temperature. The bound antibody was visualised using biotinylated secondary antibody, avidin-biotin peroxidase complex, and and 3-amino-9-ethylcarbazole. The five most vascularized areas ("hot spot") were selected at low magnification and both individual vessels were counted at x 400 magnification. In serial sections each single tryptase-positive MC was counted. Single brown stained endothelial cells and red MCs positive to tryptase were also evaluated in terms of immunostained area at x 400 magnification.Results: Our data demonstrated a significantly (r= ranging from 0.78 to 0.89; p: ranging from 0.001 to 0.002 by Pearson's analysis respectively) correlation between MCDPT, MCAPT, MVD, EA each to other, whereas no correlation concerning MCDPT, MCAPT, MVD, EA and the main clinical pathological features was found.Discussion: These preliminary results suggest that tryptase-positive MC play a key role in breast cancer angiogenesis and that inhibition of tryptase may be a new antiangiogenic strategy for the treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2159.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

UVA-induced calcium oscillations in rat mast cells

UVA is a major bio-active component in solar irradiation, and is shown to have immunomodulatory and anti-inflammatory effects. The detailed molecular mechanism of UVA action in regard to calcium signaling in mast cells, however, is not fully understood. In this study, it was found that UVA induced ROS formation and cytosolic calcium oscillations in individual rat mast cells. Exogenously added H2O2 and hypoxanthine/xanthine oxidase (HX/XOD) mimicked UVA effects on cytosolic calcium increases. Regular calcium oscillation induced by UVA irradiation was inhibited completely by the phosphatidylinositol-specific phospholipase C inhibitor U73122, but U73343 was without effect. Tetrandrine, a calcium entry blocker, or calcium-free buffer abolished UVA-induced calcium oscillations. L-type calcium channel blocker nifedipine and stores-operated calcium channel blocker SK&F96365 had no such inhibitory effect. ROS induction by UVA was abolished after pre-incubation with anti-oxidant NAC or with NAD(P)H oxidase inhibitor DPI; such treatment also made UVA-induced calcium oscillation to disappear. UVA irradiation did not increase mast cell diameter, but it made mast cell structure more granular. Spectral confocal imaging revealed that the emission spectrum of the endogenous fluorophore in single mast cell contained a sizable peak which corresponded to that of NAD(P)H. Taken together, these data suggest that UVA in rat mast cells could activate NAD(P)H oxidase, to produce ROS, which in turn activates phospholipase C signaling, to trigger regular cytosolic calcium oscillation.

Hyaluronate and its receptors in bone marrow

Cell-cell and cell-matrix interactions, which are mediated by cell adhesion molecules, play a fundamental role during many cellular processes including growth, differentiation, cell migration and cancer metastasis. One molecule playing a major role in these processes is the CD44 surface receptor, which is expressed in a wide range of cells including many cells of the hemopoietic system, where it mediates the interaction with its major ligand, hyaluronate. However, little is known about CD44 and hyaluronate in bone marrow and this was investigated immunohistochemically in trephine biopsies and in cultivated human bone marrow stromal cells. In biopsy specimens, patches of hyaluronate deposition were detected in the extracellular matrix (ECM). However, most of the areas of the ECM were devoid of hyaluronate. Single mast cells and lymphocytes scattered throughout the marrow were CD44 immunopositive. Marrow-derived stromal cells (MDSC) expanded in cell culture were immunopositive for CD44, hyaluronate synthase, and hyaluronate. Hence, a marked difference between CD44 immunolocalisation and hyaluronate deposition can be observed between in situ and under cell culture conditions. Since in normal marrow in situ the number of CD44 immunopositive cells was low, interactions of CD44 and hyaluronate would appear to not to play a major role in cell adhesion in the normal bone marrow.

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule-granule fusion before plasma membrane fusion was recorded.

Confocal Laser Scanning Microscopy to Study Molecular Mechanism of Mast Cell Activation

AbstractFor Abstract see ChemInform Abstract in Full Text.

Confocal Laser Scanning Microscopy to Study Molecular Mechanism of Mast Cell Activation

In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.

Evolution of Urticaria Pigmentosa into Indolent Systemic Mastocytosis: Abnormal Immunophenotype of Mast Cells without Evidence of c-kit Mutation ASP-816-VAL

Mastocytosis comprises a heterogeneous group of hematological disorders which are morphologically defined by proliferation and accumulation of tissue mast cells in one or more organs. Clinical manifestations of mastocytosis range from disseminated maculopapular skin lesions (=urticaria pigmentosa [UP]) that may spontaneously regress to highly aggressive neoplasms like mast cell leukemia or mast cell sarcoma. Recently, it could be shown that systemic mastocytosis (SM) is a clonal disorder often exhibiting mutations of c-kit, a protooncogene encoding the tyrosine kinase receptor for stem cell factor (SCF). Mutations of c-kit are considered to play a key role in the pathogenesis of mastocytosis. Therefore, we investigated the unique case of a 36 year-old male patient with indolent systemic mastocytosis (ISM) evolving from UP (cutaneous mastocytosis) by means of histology, immunophenotyping and molecular biology. At the time of initial diagnosis the bone marrow showed only a mild diffuse increase in mast cells but compact infiltrates were missing. The serum tryptase levels were normal. Five years later, however, the bone marrow histology displayed patchy compact mast cell infiltrates, which now allowed to establish the diagnosis of an ISM. The serum tryptase levels at this time were markedly elevated. At both time points, mast cells were analyzed by immunohistochemistry using anti-tryptase antibody AA1, by flow cytometry using antibodies against CD2 and CD25, and nested polymerase chain reaction (PCR) on laser-microdissected, single pooled mast cells. Immunohistochemistry revealed strong tryptase-positivity of mast cells in both cutaneous and bone marrow infiltrates. Flow cytometry yielded an aberrant expression of CD2 and CD25 on bone marrow mast cells. However, repeated thorough PCR analysis failed to unveil c-kit mutation in atypical mast cells of skin and bone marrow samples of both dates. These findings clearly show that ISM can evolve from UP. Moreover, our study provides further evidence that the c-kit mutation Asp-816-Val is not invariably present in ISM.

Detection of c- kit point mutation Asp-816 → Val in microdissected pooled single mast cells and leukemic cells in a patient with systemic mastocytosis and concomitant chronic myelomonocytic leukemia

The c- kit mutation Asp-816→Val is detectable not only in neoplastic mast cells (MCs) in patients with systemic mastocytosis (SM) but also in most associated hematologic non-MC lineage disease (AHNMD). In order to prove a monoclonal disease evolution we investigated DNA of pooled microdissected single cells for the presence of the mutation in a patient with SM and concomitant chronic myelomonocytic leukemia (CMML). LightCycler melting curve analysis and direct sequencing of nested polymerase chain reaction (PCR) products revealed the c- kit mutation in tryptase-positive MC and in leukemic CD15-positive cells in bone marrow infiltrates, but not in colonic epithelial cells, thus, suggesting a monoclonal evolution of SM and concurrent CMML on the basis of a somatic mutation in a common hematologic progenitor.

Amperometric characterization of exocytotic events from single mast cells: dependence on external and internal Ca++sources

Mast cells exocytotically release histamine/serotonin in response to different secretagogues. We have used substance P and compound 48/80 to study the Ca++dependency of serotonin exocytosis from peritoneal mast cells using carbon fiber amperometric techniques. The exocytotic release pattern consists of a burst of events superimposed on a slow, transient, amperometric current baseline increase. Cellular re lease parameters (number, frequency and total charge of amperometric events) and individual event characteristics (charge integral, half width and peak amplitude) were similar for the two secretagogues used. Zero Ca++conditions greatly reduced, without completely abolishing,cellular release parameters. Cyclopiazonic acid, an inhibitor of the endoplasmatic Ca++ATPase, reduced the cellular exocytotic capacity and diminished the amplitude of individual exocytotic events more effectively than the 0 Ca++condition. The cyclopiazonic acid effects occurred in the presence of external Ca++, indicating that this condition is not sufficient for maintaining full exocytotic capacity. The results confirm the importance of intracellular Ca++for exocytotic activation. For the first time evidence is presented that the integrity of intracellular Ca++pools determines the amplitude and frequency of individual exocytotic events. Saponin, a non-specific detergent, also induced quantal release similar to that obtained with substance P and compound 48/80. This release was not dependent on extracellular C a++, but cyclopiazonic acid significantly reduced individual exocytotic release.

Direct detection of endogenous histamine in rat peritoneal mast cells by in-capillary derivatization high-performance capillary electrophoresis

A simple method for the detection of endogenous histamine in rat peritoneal mast cells was evaluated using on-line mode in-capillary derivatization high-performance capillary electrophoretic (ICD-HPCE) techniques, which were previously developed by our group [S. Oguri et al., J. Chromatogr. A, 787 (1997) 253–260]. The method involves a suspension of peritoneal mast cells (1·10 6 cells/ml of saline) collected from a male Wistar rat (eight weeks of age), which are directly introduced into the capillary tube from the anodic end by hydrostatic injection (at 25 cm height, for 2–20 s). When a high-voltage potential (25 kV) is applied to the capillary, which is already filled with the run buffer containing both a lysing reagent (SDS, sodium dodecyl sulfate,) and a derivatizing reagent (OPA, o-phthalaldehyde; NAC, N-acetylcysteine), histamine in the mast cells was detected at high-sensitivity level without further procedures. During ICD-HPCE, the mast cells injected in the capillary were lysed with the lysing reagent, free histamine released from the cell was labeled with the derivatizing reagent, and its derivative was electromigrated, separated and detected with a fluorescence detector (excitation wavelength at 340 nm, emission wavelength at 450 nm) in a fused-silica capillary (75 cm effective length×50 μm I.D.). The run buffer used was a 20 m M phosphate–borate buffer (pH 10) containing 20 m M SDS, 2 m M OPA and 2 m M NAC. This method was also examined with regard to the possibility of its use for determination of histamine at the single mast cell level.