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Flow Cytometric Detection (flow + cytometric_detection)
Selected AbstractsSimultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy,CYTOMETRY, Issue 1 2009Nicolaas E. Aerts Abstract Background: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. Methods: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. Results: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 ,g/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. Conclusion: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation. © 2008 Clinical Cytometry Society [source] Human B cells express a CD45 isoform that is similar to murine B220 and is downregulated with acquisition of the memory B-cell marker CD27,CYTOMETRY, Issue 1 2003Jack J. H. Bleesing Abstract Background Differences between human and murine B cells exist at all stages of B-cell development, including the stage of memory B-cell formation. B cells in mice are identified with the pan,B-cell,specific CD45 isoform, B220. In initial studies in humans, it appeared that B220 expression did not include all B cells. This study was performed to expand on those preliminary findings. Methods Multiparameter flow cytometric detection of B220 expression on B cells was combined with a variety of B-cell markers. Results In contrast to mice, B220 was not a pan,B-cell marker in humans but was downregulated in the majority of B cells that acquired the human memory B-cell marker, CD27, whereas a minor memory B-cell subset remained B220+, suggesting differences in differentiation. Conclusions The B220 isoform in humans is developmentally regulated in humans, tied to the acquisition of a memory phenotype, and as such can be used as a differentiation-specific CD45 isoform, akin to the use of CD45 isoforms to distinguish between naive and memory T-cell subsets. Patients with immunodeficiency disorders, associated with defective memory B-cell generation and absent or reduced CD27+ B cells, showed a corresponding lack of B220 downregulation consistent with altered differentiation of B-cell subsets. Cytometry Part B (Clin. Cytometry) 51B:1,8, 2003. Published 2002 Wiley-Liss, Inc. [source] Dynamics of marine bacterial and phytoplankton populations using multiplex liquid bead array technologyENVIRONMENTAL MICROBIOLOGY, Issue 4 2010Xavier Mayali Summary Heterotrophic bacteria and phytoplankton dominate the biomass and play major roles in the biogeochemical cycles of the surface ocean. Here, we designed and tested a fast, high-throughput and multiplexed hybridization-based assay to detect populations of marine heterotrophic bacteria and phytoplankton based on their small subunit ribosomal DNA sequences. The assay is based on established liquid bead array technology, an approach that is gaining acceptance in biomedical research but remains underutilized in ecology. End-labelled PCR products are hybridized to taxon-specific oligonucleotide probes attached to fluorescently coded beads followed by flow cytometric detection. We used ribosomal DNA environmental clone libraries (a total of 450 clones) and cultured isolates to design and test 26 bacterial and 10 eukaryotic probes specific to various ribotypes and genera of heterotrophic bacteria and eukaryotic phytoplankton. Pure environmental clones or cultures were used as controls and demonstrated specificity of the probes to their target taxa. The quantitative nature of the assay was demonstrated by a significant relationship between the number of target molecules and fluorescence signal. Clone library sequencing and bead array fluorescence from the same sample provided consistent results. We then applied the assay to a 37-day time series of coastal surface seawater samples from the Southern California Bight to examine the temporal dynamics of microbial communities on the scale of days to weeks. As expected, several bacterial phylotypes were positively correlated with total bacterial abundances and chlorophyll a concentrations, but others were negatively correlated. Bacterial taxa belonging to the same broad taxonomic groups did not necessarily correlate with one another, confirming recent results suggesting that inferring ecological role from broad taxonomic identity may not always be accurate. [source] rRNA PROBES FOR IDENTIFICATION AND CHARACTERIZATION OF MARINE PHYTOPLANKTON: THEIR POTENTIAL APPLICATION FOR DNA MICROCHIPSJOURNAL OF PHYCOLOGY, Issue 2001Article first published online: 24 SEP 200 Groben R., Lange, M. & Medlin, L. K. Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany A fast and reliable identification of nano- and picoplankton by light microscopy is often difficult because of the lack of usable morphological characteristics, whereas electron microscopy and biochemical methods are very time consuming. Identification of toxic algae also requires a great deal of taxonomic experrtise so that false positives are not recorded. One solution is to use taxon specific rRNA probes. For this purpose we designed probes for phytoplankton taxa, including toxic algae. These probes were either labelled with Digoxigenin (DIG) and used in DNA dot blot experiments, or labelled with fluorochromes and used in whole-cell hybridisations with fluorescence microscopy or flow cytometric detection. Specific probes could be used over a broad taxonomic range from higher groups (i.e. the class of dinoflagellates) to species level (i.e. Prorocentrum lima). These probes were be used in the EU MAST project AIMS for the development of an automated identification system for marine phytoplankton in combination with flow cytometry and artificial neural networks (ANNs), in the EU MAST DETAL and in the German national project (TEPS) for the development of an early warning system for harmful algal blooms. Results using Digoxigenin (DIG)-labelled probes on picoplankton samples taken from several water bodies indicate that hierarchial re-probing of spotted samples can be achieved and this suggests that probes can be adapted to DNA microchips. Preliminary field results for a hand-held DNA microchip reader are presented. This work was supported by the German BMBF TEPS 03F0161 and the EU AIMS MAS3-CT97-0080 and EU DETAL Q5RS-2000-30778 projects. [source] Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemiaAMERICAN JOURNAL OF HEMATOLOGY, Issue 2 2001Rogelio Paredes-Aguilera Abstract To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study. Am. J. Hematol. 68:69,74, 2001. © 2001 Wiley-Liss, Inc. [source] Flow cytometric analysis of BCL-2 can distinguish small numbers of acute lymphoblastic leukaemia cells from B-cell precursorsBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2004Leah Hartung Summary Flow cytometric identification of small numbers of precursor B-cell acute lymphoblastic leukaemia (B-ALL) cells in post-treatment marrow specimens could benefit from the identification of additional, easily detectable markers that could be used in most cases. In this study, we evaluate whether bcl-2 expression quantified by four-colour flow cytometry can be effectively used to discriminate precursor B-ALL blasts from normal B-cell precursors (haematogones) and function as a leukaemia-specific marker. Levels of bcl-2 in the 22 precursor B-ALL cases studied were found to be significantly higher (over sixfold higher on average) than those present in haematogone populations from 22 control marrow specimens. Higher relative levels of bcl-2 expression in the B-ALL cases were maintained with respect to both immature CD34+ and more mature CD34, haematogone subsets, and appeared stable. Dilutional studies indicated that multiparameter flow cytometry analysis using bcl-2 could identify precursor B-ALL blasts representing as few as 1% of CD19+ cells or 0·01% of total leucocytes in bone marrow specimens containing substantial numbers of haematogones. This study suggests that bcl-2 may be an important marker for flow cytometric detection and quantification of small numbers of residual precursor B-ALL cells in bone marrow specimens. [source] | |||||||||||||