Phagocytosis: measurement by flow cytometry

Lehmann, Anne Kristine; Sørnes, Steinar; Halstensen, Alfred

doi:10.1016/s0022-1759(00)00237-4

Cited by 162 publications

(137 citation statements)

References 55 publications

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“…This biological behavior seems to characterize GPI-defective PMN in PNH, if compared with neutropenic or CGD patients. Indeed, our data suggest that the complete defect in ROS production is not accompanied by significant increased ingestion ability in CG, according to previous results [53,54]. On the basis of this observation, one could rule-out that increased engulfment could represent only a compensatory mechanism for the deficient burst induction.…”

Section: Discussionsupporting

confidence: 88%

Glycosyl‐phosphatidyl‐inositol‐defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction

et al. 2006

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Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the emergence of a GPI-defective clonal hematopoiesis. Its clinical features are hemolytic anemia, cytopenia, and thrombosis. Circulating monocytes and granulocytes are largely GPI-defective in PNH patients. This study aims to investigate the granulocyte functional properties in PNH. We analyzed bacterial-dependent intracellular ingestion and the consequent activation of oxidative burst in GPI-defective granulocytes from four neutropenic PNH patients. Our data show a significant increase in the ability of GPI-defective granulocytes to ingest opsonized bacteria. In addition, an impaired respiratory burst effectiveness in response to two independent bacterial stimuli, the N-formyl-MetLeuPhe (fMLP) synthetic bacterial peptide and E. coli, was revealed. The occurrence of neutropenia and the severe impairment of oxidative burst, occurring in chronic granulomatosis disease, were unable to significantly affect phagocytosis. Thus, additional mechanisms, able to differentially affect ingestion ability and respiratory burst effectiveness, have to be hypothesized. The reduced burst effectiveness of GPIdefective granulocytes was maintained after treatment with phorbol 12-myristate 13-acetate, a pharmacological stimulus able to extensively recruit and to trigger intracellular protein kinase C (PKC). Moreover, blocking of PKC has been observed to severely affect granulocyte respiratory burst with a mild effect on the phagocytosis. These data suggest a role for a modulation of intracellular PKC in the pathogenesis of the impaired granulocyte oxidative burst. Am. J. Hematol. 82:98-107, 2007. V V C 2006 Wiley-Liss, Inc.

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Section: Discussionsupporting

confidence: 88%

Glycosyl‐phosphatidyl‐inositol‐defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction

et al. 2006

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“…The presence of hydrogen peroxide was detected by oxidation of dichlorfluorescein diacetate (DCFH-DA; Sigma-Aldrich) into green fluorescent dichlorfluorescein (21). The phagocytic activity was evaluated by ingestion of PE-labeled polystyrene micro spheres (1 m diameter, Fluoresbrite Plain Microspheres PCRed; Polysciences, Warrington, PA) as described previously (6).…”

Section: Detection Of Respiratory Burst and Phagocytosismentioning

confidence: 99%

“…Since TREM-1 ligation results in rapid degranulation, we investigated whether other PMN effector functions are also involved in TREM-1-mediated activation. We determined the effect of TREM-1 stimulation on respiratory burst and phagocytosis by analysis of hydrogen peroxide production and phagocytosis of unopsonized polystyrene beads, as described previously (6,21). As shown in Fig.…”

Section: Trem-1 Mediates Respiratory Burst and Phagocytosismentioning

confidence: 99%

Triggering Receptor Expressed on Myeloid Cells-1 in Neutrophil Inflammatory Responses: Differential Regulation of Activation and Survival

Radsak

Salih

Rammensee

et al. 2004

The Journal of Immunology

184

176

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Polymorphonuclear neutrophils (PMN) are crucial in the innate host defense by their ability to rapidly accumulate in inflamed tissues and clear a site of infection from microbial pathogens by their potent effector mechanisms. The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described activating receptor on PMN with an important role in inflammation. However, the effects of TREM-1 stimulation on a cellular level remain to be further defined. To characterize TREM-1-mediated activation of human PMN, we evaluated the effect of receptor ligation on PMN effector functions. Activation via TREM-1 induces immediate degranulation of neutrophilic granules resulting in the release of IL-8, respiratory burst, and phagocytosis. TREM-1 ligation synergizes with the activation by the Toll-like receptors (TLR) ligands LPS, Pam3Cys, and R-848. In contrast, no synergy between TREM-1- and TLR-mediated stimulation was observed concerning PMN survival, whereas TLR-mediated stimuli protect PMN from apoptosis, concurrent TREM-1 activation neutralizes these anti-apoptotic effects. These results give a new perspective for the regulation of neutrophil inflammatory responses emphasizing the importance of TREM-1 in innate immunity.

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“…The most frequently used particles in phagocytotic measurements are zymosan (1)(2)(3)(4)(5) and Staphylococcus aureus (1,2,6-9), labeled almost without exception with fluorescein isothiocyanate (FITC). FITC-labeled Streptococcus pneumoniae (10,11), Escherichia coli (12), Neisseria meningitis (13), latex particles (5,12,14,15), herpes simplex virus (16,17), and entire yeasts such as Saccharomyces cerevisiae (18)(19)(20), and Candida albicans (8,21,22) have also been used. In this study, zymosan was used as a phagocytosable particle.…”

mentioning

confidence: 99%

“…Phagocytotic methods presented in literature are frequently characterized by a high phagocytotic concentration in a low reaction volume (usually 2-5 3 10 6 /ml in 0.1-0.25 ml) (5,9,13,15,17,24,26,27). Another distinctive feature of many phagocytotic methods presented in the literature is that they often use only a single measuring time point instead of kinetic measurement (4)(5)(6)(7)10,11,13,17,(20)(21)(22)24,28,29). Opsonization of phagocytosable particles has been performed without exception by adding serum or plasma (5-20%) into a reaction mixture.…”

mentioning

confidence: 99%

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells

2005

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Background: The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)-labeled zymosan particles by human leukocytes. Methods: FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence. Results: The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equa-

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Phagocytosis: measurement by flow cytometry

Cited by 162 publications

References 55 publications

Glycosyl‐phosphatidyl‐inositol‐defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction

Glycosyl‐phosphatidyl‐inositol‐defective granulocytes from paroxysmal nocturnal haemoglobinuria patients show increased bacterial ingestion but reduced respiratory burst induction

Triggering Receptor Expressed on Myeloid Cells-1 in Neutrophil Inflammatory Responses: Differential Regulation of Activation and Survival

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells

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