Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Single cell”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed2006

Dynamic compression of single cells.

OBJECTIVE: The objective of this study was to measure the effects of dynamic compression on single chondrocyte gene expression using a single cell approach, combining single cell biomechanics with single cell gene expression. DESIGN: Articular chondrocytes from the middle and deep zones of bovine distal metatarsal cartilage were statically or dynamically compressed (at a frequency of approximately 1Hz) using a custom creep cytocompression apparatus, and their gene expression levels for type II collagen, aggrecan, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1 were subsequently measured using single cell real-time reverse transcriptase-polymerase chain reaction. RESULTS: Single chondrocyte gene expression was lognormally distributed, suggesting that studies of populations of cells may be biased by a minority of cells with very high levels of gene expression, and would not accurately describe the behavior of most chondrocytes. Chondrocytes exposed to dynamic loading did, in general, have higher levels of type II collagen and aggrecan gene expression than statically loaded cells. Specifically, compressive forces of 50 and 100 nN suppressed type II collagen expression when applied statically, but the equivalent dynamic loads increased expression to control levels. Tissue inhibitor of metalloproteinase-1 was not affected by the mechanical loading regimens examined. CONCLUSIONS: We have demonstrated that a single cell approach is a viable methodology for studying the responses of cells to mechanical forces. Furthermore, examining the effects of mechanical loading on a cell-by-cell basis allows us to capture behaviors and details that would otherwise elude studies performed on a larger scale.

Aggrecans↗
PubMed1996

Classical Hodgkin and Reed-Sternberg cells demonstrate a non-clonal immature B lymphoid lineage: evidence from a single cell assay and in situ hybridization.

Hodgkin and Reed-Sternberg (H & RS) cells are generally considered to be the neoplastic cells of Hodgkin's disease (HD), however such cells are only found in a minority of the lesions. Recently in a few studies on HD, the clonality of H & RS cells was examined, using a single-cell polymerase chain reaction (PCR) examination. To clarify the lineage and clonality of H & RS cells, we performed single cell PCR and in situ hybridization (ISH), and nine cases of classical HD were thus studied. By ISH, the immunoglobulin J chain, and the kappa and lambda light chain were rarely expressed in the H & RS cells, however, no T-cell markers could be detected. The expression of the recombination activating genes (RAG-1, 2) could be determined in the H & RS cells. We isolated CD30+ H & RS cells, CD3 + T cells and CD20 + B cells from suspended materials using a mechanical sorter. We performed single cell PCR in a sorted individual cell, to amplify the complementarity determining region of the Ig heavy chain (IgH) gene and T-cell receptor gamma chain (TCR gamma) gene. In all cases, TCR gamma could be frequently amplified in the T cells, but was only rarely amplified in the H & RS and B cells. In contrast, the IgH was frequently amplified in the H & RS and B cells, but not in the T cells. In addition, the PCR production of the H & RS cells all showed different lengths. The results therefore support the polyclonal nature and immature B lymphoid cell origin of H & RS cells.

B-Lymphocyte Subsets↗
PubMed2003

Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis.

Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR beta chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.

Adult↗
PubMedDate not supplied

Genotoxicity of chromium in human gastric mucosa cells and peripheral blood lymphocytes evaluated by the single cell gel electrophoresis (comet assay).

Hexavalent chromium compounds are well-recognized carcinogens. They easily penetrate the cell membrane and are reduced inside the cell to their trivalent form, which is supposed to react directly with DNA. Chromium is present in some workplaces as well as in water resources and food chain, so it can interact with the mucosa of the gastrointestinal tract. In order to elucidate the genotoxic potency of chromium in human gastric mucosa (GM) cells, the DNA-damaging effect of potassium dichromate (K2Cr2O7) was investigated using alkaline single cell gel electrophoresis (comet assay). Biopsy samples were obtained during gastroscopy from macroscopically healthy tissue of the stomach. Parallel test with human peripheral blood lymphocytes was also performed. Both types of cells were incubated at 37 degrees C with 1.6 mM of K2Cr2O7 for 1 h and after washing, were placed in a chromium-free medium to examine DNA repair. Alkaline single cell gel electrophoresis (comet assay) was used to assess DNA damage and repair. Chromium introduced a damage to DNA both in the GM cells and lymphocytes. The effect induced by K2Cr2O7 in GM cells was comparable with that caused in the lymphocytes. Treated cells were able to recover within a 60-min incubation in a chromium-free medium at 37 degrees C. The results obtained indicate that hexavalent chromium compounds, which may be found in the diet, can interact directly with DNA of the mucosa of the stomach.

Comet Assay↗
PubMedDate not supplied

On-chip single-cell-based microcultivation method for analysis of genetic information and epigenetic correlation of cells.

We have developed an on-chip single-cell based microcultivation method for analyzing the variability of genetic information stored in single cells and their epigenetic correlations. The method uses four systems: an on-chip cell sorter for purifying the cells in a non-destructive manner; an on-chip single-cell cultivation chip for isolating single cells; an on-chip agarose microchamber system for constructive cell-cell network formation during cultivation; and an on-chip single-cell-based expression analysis system. Using these systems, we could measure the variability of prokaryotic cells and eukaryotic cells having the same DNA and found that, although prokaryotic cells have a large variability in their interdivision times, sister eukaryotic cells having the same DNA synchronized well. We also measured the dynamics of synchronization of beating cardiac myocytes and found that two isolated cells synchronize by one cell following the other after a short pause in beating. These results showed the potential of the on-chip microcultivation method's constructive approach to analyzing cell systems.

Animals↗
PubMed1980

Single-cell immunofluorescence assay for terminal transferase: human leukaemic and non-leukaemic cells.

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT(+) cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT(+) cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin's disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT(+) cells were also found in association with cells with lymphoblast morphology. The TdT(+) cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT(+) cells at diagnosis, the percentage TdT(+) cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT(+) cells at diagnosis also showed < 1% TdT(+) cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in a conventional biochemical assay, is highly specific, and could provide a technically more efficient alternative for use in clinics as well as in experimental investigations of subpopulations of leukaemic and normal marrow cells.

Bone Marrow↗
PubMed2003

Expansion and proliferation of skin-homing T cells in atopic dermatitis as assessed at the single cell level.

BACKGROUND: Sensitization to aeroallergens is a frequent event in patients with atopic dermatitis (AD). The role of allergen-specific T cells in the pathogenesis of AD is, however, not fully clear. A detailed immunological characterization of allergen-specific T cells able to migrate to inflamed skin might therefore be helpful in determining the relevance of allergen-specific T cells to clinical symptoms. OBJECTIVE: To establish a simple assay to simultaneously monitor the expression of skin-related homing molecules and their proliferative response to an allergen on peripheral T cells. To evaluate whether this assay is able to identify AD patients in whom exposure to an allergen induces dermatitis. METHODS: The expression of CLA, CD7 and CD4 was simultaneously measured by flow cytometry with the DNA content as a parameter of proliferation at the single cell level. House dust mite antigen (DerP) and tetanus toxoid (TT) were used as antigens. RESULTS: CD4+ CLA+ CD7-, but not CLA- T cells from patients with AD proliferated and expanded following DerP stimulation. In contrast, TT stimulation resulted in proliferation of both, CLA+ and CLA- T cells from both, atopic and healthy donors. CONCLUSIONS: Single cell analysis revealed a predominant localization of allergen-reactive T cells within the CD7- CLA+ CD4+ T cell subset in AD. Thus, this simple assay not only confirms previously published results, but also extends our knowledge on the biology of CLA and CD7 cells in AD. Ultimately, this will be helpful in classifying those patients who might benefit from allergen avoidance therapies.

Adult↗
PubMedDate not supplied

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell.

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.

Cell Division↗
PubMed1996

The use of the single cell gel electrophoresis assay in detecting DNA single strand breaks in lung cells in vitro.

DNA single strand breaks (SSB) can be used as a biomarker of oxidant exposure, and also as an indicator of the carcinogenicity/ mutagenicity of a substance. The single cell gel electrophoresis (SCGE) assay is more sensitive and requires fewer cells compared to other techniques used for detecting SSB. We examined the utility of using the SCGE assay for human lung cells exposed to endogenous and exogenous oxidants. A human bronchial cell line (BEAS) was used as a model of airway epithelial cells in this study. BEAS cells exposed to 0-50 microM hydrogen peroxide (H2O2) for 60 min at 4 degrees C exhibited a concentration-dependent increase in SSB as determined by an increased DNA migration area in a gel undergoing electrophoresis. H2O2-induced increases in DNA SSB were also demonstrated using cultured normal human tracheobronchial epithelial (NHBE) cells and human alveolar macrophages in a concentration response manner. BEAS cells were also exposed to air or ozone (O3) on a Transwell filter without medium present apically. Cells exposed to O3 at 0.1 or 0.4 ppm at 37 degrees C for up 120 min had a time- and concentration-dependent increase in SSB compared to air-exposed cells. NHBE cells exposed to 0.4 ppm O3 (60 min) also had increased DNA SSB. Cells with H2O2-induced DNA SSB can be frozen and stored up to 4 weeks without altering the original DNA SSB. These findings indicate that SCGE can be used to detect SSB in cultured lung cells, and has applicability for detecting SSB in lung cells recovered from in vivo and in vitro exposures to oxidants.

Bronchi↗
PubMedDate not supplied

A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

Animals↗
PubMed1997

Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human luteinizing-hormone receptor.

We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca2+]i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH beta- or alpha-subunits, evoked specific [Ca2+]i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca2+]i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca2+]i responses were observed in 12-14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca2+]i signal amplitude), and eliciting a [Ca2+]i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca2+]i spike-and-plateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn2+ following the [Ca2+]i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca2+]i response, and pertussis toxin (PTX) did not alter LH-stimulated [Ca2+]i signaling. However, overnight preincubation with LH reduced the percentage of [Ca2+]i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca2+]i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca2+ signaling response by recruiting an increasing number of [Ca2+]i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca2+]i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca2+]i signal.

Adenylate Cyclase Toxin↗
PubMed2006

Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays.

High-quality single-cell data are required for a quantitative systems biology description of cellular function. However, data of this type are difficult and time-consuming to collect using traditional techniques. We present a robust and simple microfluidic method for trapping single cells in large arrays to address this problem. Ordered single-cell isolation arrays allow for high-density microscopic analysis with simplified image processing. Moreover, for fluorescent assays, on-chip sample preparation (e.g., fluorescent labeling, washing) can be performed, as opposed to manual intensive operations of incubation, centrifugation, and resuspension in previous techniques-saving time and reagents. This technology was applied to determine novel single-cell enzyme kinetics for three different cell types (HeLa, 293T, Jurkat). A kinetic model of this process predicted this varied response was due to variation in the concentration of carboxylesterase between cell types. Nordihydroguaiaretic acid (NDGA) was also characterized as an inhibitor of carboxylesterases. For HeLa cells, 20 nM of the 50 nM total carboxylesterases was unaffected by NDGA. This type of analysis could be directly applied to quantify a variety of intracellular enzymes with available fluorogenic substrates.

Carboxylesterase↗
PubMed1994

Sequential fates in a single cell are established by the neurogenic cascade in the Malpighian tubules of Drosophila.

In each Malpighian tubule of Drosophila, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. We follow the segregation of this cell, explore the genetic interactions that underlie its specification and demonstrate that tip cell allocation closely resembles neurogenesis. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells in each tubule primordium. Each cluster is marked out by the expression of proneural genes and the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. We confirm the mitogenic role of the tip cell during embryogenesis by mutational analysis and show that it subsequently adopts a second fate, differentiating neural characteristics. We demonstrate that both stages in the differentiation of this cell are established by the same sequence of genetic interactions, which have not previously been shown to occur outside the neurogenic ectoderm.

Animals↗
PubMed1996

Radiotoxic effect and dosimetry of 67GA in multicellular spheroids as compared to single cells of the lymphoma cell line U715.

PURPOSE: The purpose of the present study was to investigate if there were differences between U715 spheroids and single cells in the radiotoxic effect of 67Ga on cell growth and clonogenic capacity in vitro and to generate dosimetric approaches for the multicellular tumor model. METHODS AND MATERIALS: Human lymphoma U715 cells were cultured in vitro as single cells and multicellular spheroids, grown with the use of a combination of fibrin clot technique, spinner flasks, and liquid-overlay culture. Cells were incubated with 2.96-8.88 MBq/ml 67Gallium for 4 days. Spheroids were dispersed to single cells by treatment with plasmin. Residual proliferative and clonogenic capacity after 67Ga incubation were assayed using the MTT-test and clonogenic test, respectively. Autoradiography was performed with 1 microm sections and Ilford L4 liquid photographic emulsion. Dosimetric approaches were made, based on the MIRD-approach. RESULTS: During 67Ga incubation proliferation was inhibited. The residual proliferative or clonogenic capacity was inhibited by 8.88 MBq/ml for 39 and 88%, respectively. For single cells with 6.66 MBq/ml these inhibitions were 64 and 96%, respectively. Autoradiography showed an homogeneous distribution of 67Ga in spheroids and single cells. In single cells a 2.1-3.5 times higher 67Ga uptake/cell than in spheroids produced an equitoxic effect. The uptake parameters were implemented in new dosimetric approaches and showed that the efficacy of intracellular 67Ga was two times higher in spheroid clusters than in single cells due to energy deposition of internal conversion electrons within the cell clusters with a mean diameter size of nine cells. Both for proliferative and clonogenic capacity the exponential survival curves were superimposed. CONCLUSIONS: With the new approaches made in our dosimetric model the discrepancy found between 67Ga accumulation and radiotoxic effect in spheroids as compared to single cells can be explained by additional effects of the crossfire of internal conversion electrons within clusters of about nine cells in diameter in spheroids. Only twice as much 67Ga was needed to reach equitoxic absorbed doses in spheroids than was needed in single cells. Such might be important for the use of 67Ga treatment of small metastasis of malignant lymphoma.

Autoradiography↗
PubMed2000

Single-cell analysis avoids sample processing bias.

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

Electrophoresis, Capillary↗
PubMed1996

Increasing the denaturation temperature during the first cycles of amplification reduces allele dropout from single cells for preimplantation genetic diagnosis.

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) needs to be highly efficient and accurate. In some single cells from human embryos presumed to be heterozygous for the delta F508 deletion causing cystic fibrosis (CF), we recently observed random amplification failure of one of the two parental alleles following nested PCR. To investigate allele dropout (ADO), we have examined two different lysis protocols and the effect of altering the denaturation temperature in the primary PCR using single lymphocytes heterozygous for delta F508 or for two beta-thalassaemia mutations IVS 1 nt 1 (G/T) and 5 (G/C) using a nested PCR protocol to amplify the 5' region of the beta-globin gene. Amplification rates were high after lysis in either water or lysis buffer and at all denaturation temperatures studied (> or = 92%). With a typical denaturation temperature (93 degrees C), ADO was detected at both loci. When the denaturation temperature was lowered to 90 degrees C, however, ADO increased substantially and conversely by raising the denaturation temperature to 96 degrees C during the first 10 cycles ADO was reduced but not eliminated. ADO was also reduced with cells in lysis buffer. We suggest that ADO may be caused by a combination of inefficient denaturation and degradation of one of the genomic alleles in the first cycles of PCR. For autosomal recessive conditions in which both parents are carrying the same mutation, ADO would not cause serious misdiagnosis. For compound heterozygotes or autosomal dominant conditions, however, extensive testing of the amplification protocol with single heterozygous cells and individual calibration of each thermocycler for the effect of denaturation temperature on ADO is essential before clinical application.

Alleles↗
PubMed2003

A method of analyzing procoagulant activity in monocytes at single cell level.

BACKGROUND AND OBJECTIVES: Procoagulant activity (PCA) of monocytes is known to play a pivotal role in a variety of physiologic and pathophysiologic processes, such as disseminated intravascular coagulation, atherosclerosis, arterial and venous thromboembolism, cancer-related hypercoagulability and immunopathologies. Until now, PCA has been studied by clotting assays of a whole cell population or at single cell level by analyzing tissue factor antigen, the protein that initiates PCA but does not always correlate with it. Here, we describe a new simple flow cytometric method that allows the PCA of monocytes to be studied at a single cell level by quantifying the fibrin formed around the cells in suspension. DESIGN AND METHODS: Purified fibrinogen was tagged with FITC and added to a recalcified developer plasma containing suitable amounts of heparin in order to inhibit the expansion of clotting, thus limiting the formation of fibrin to the surface of cells with PCA. With appropiate amounts of heparin, in 10 min, large sea urchin-like cells with fibrin needles around some monocytes were formed and, after fixation, cytofluorimetrically analyzed. RESULTS: Blood mononuclear cells isolated and immediately analyzed showed less than 0.1% sea urchin cells. Adherence alone, lipopolysaccharides or ionomycin stimulated expression of PCA in a dose- and time-dependent relationship: after 30 min, 1-3% of the MNC showed PCA, and after 20 h this reached 5-10%. Density separation of monocytes showed that different stimulators act on different maturation stages. Subjects with diabetes express more monocytes with PCA than normal subjects after 30 min stimulation. INTERPRETATION AND CONCLUSIONS: This method allows PCA analysis of monocytes at single cell level and requires only a low number of cells. The signal produced by the fluorescent fibrin is strong and easily analyzed by flow cytometry. The method is suitable for analyzing blood from patients with different pathologies and many conditions under different stimuli.

Blood Coagulation↗
PubMed1990

Measurement of cytokine release by human cells. A quantitative analysis at the single cell level using the reverse haemolytic plaque assay.

The reverse haemolytic plaque assay has been adapted to detect and measure the release of such cytokines as interleukin-1, -2 and -6, GM colony-stimulating factor or interferon-gamma by individual human cells derived from either peripheral blood or enzymatically dispersed breast carcinomas. Since each of these peptides is released by more than one cell type, this in vitro assay has been coupled with immunocytochemistry to identify the particular cell type(s) contributing to the release of each cytokine. This technique is useful in (i) obviating the need for purification of a given cell type prior to estimating cytokine release, and (ii) evaluating quantitative differences in secretion amongst cells of a particular type. Such a method has the additional advantage over most alternative methods applied at the single cell level in that the cells remain viable at the end of the assay and can be used in further studies. This assay thus provides a powerful new tool in the investigation of the role of cytokines in both the normal modulation of the immune system and the development of such diseases as neoplasia.

Biological Factors↗