Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes

Walrand, Stéphane; Valeix, Sophie; Rodriguez, Carmélita; Ligot, Philippe; Chassagne, J; Vasson, Marie‐Paule

doi:10.1016/s0009-8981(03)00086-x

Cited by 187 publications

(132 citation statements)

References 30 publications

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“…Since rhodamine 123 is known to bind to cellular and mitochondrial membranes, the fluorescent signal is mainly localized inside the cell. DHR is specifically responsive to H 2 O 2 accumulation (20). Hydroethidine (HE) is also used to characterize ROSs production inside phagocytes (21,22).…”

Section: Measurement Of Pmn Oxidative Burstmentioning

confidence: 99%

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations

Elbim

Lizard

2009

Cytometry Pt A

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Flow cytometric analysis provides a rapid screen for abnormalities of polymorphonuclear neutrophils (PMN) function and reflect their behavior in vivo more accurately. This review summarizes the major fluorescent probes used to study PMN oxidative burst and apoptosis using flow cytometry (FCM). We also provide examples of FCM studies in physiological and pathological situations, illustrating the advantages of FCM for assessment of PMN oxidative burst and PMN apoptosis. These data point to the role of FCM in detecting primary immunodeficiencies such as IRAK4 deficiency and support the use of the assessment of the PMN oxidative burst for routine testing in patients with bacterial infections. We also demonstrate the utility of whole-blood analysis using FCM for a better understanding of PMN functionality, i.e., tuning PMN responses to inflammatory stimuli. Finally, FCM permits a simultaneous analysis of phenotypic, functional and morphometric parameters assessing whole-blood PMN apoptosis, in particular in response to Toll-like receptor agonists and during simian immunodeficiency virus infection. '

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Section: Measurement Of Pmn Oxidative Burstmentioning

confidence: 99%

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations

Elbim

Lizard

2009

Cytometry Pt A

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“…Peritoneal neutrophil activation status was measured by flow cytometry using PE-labeled anti-Gr-1 Ab to identify neutrophils, and APC-labeled anti-CD11b Ab, a component of the Mac-1 complex (CD11b/CD18), was used to detect neutrophil activation. Neutrophil NADPH oxidase activity was determined using the flow cytometric-based dihydrorhodamine 123 (DHR123) assay as described by Walrand et al (19). In brief, peritoneal lavage cells (5 ϫ 10 5 cells/well of a 96-well plate) were incubated for 25 min at 37°C in the presence or absence of 4 g/ml DHR123.…”

Section: Measurement Of Peritoneal Neutrophil Activation Status and Pmentioning

confidence: 99%

Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function

Maung

Fujimi²,

MacConmara³

et al. 2008

The Journal of Immunology

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Major injury is widely thought to predispose the injured host to opportunistic infections. This idea is supported by animal studies showing that major injury causes reduced resistance to polymicrobial sepsis induced by cecal ligation and puncture. Although cecal ligation and puncture represents a clinically relevant sepsis model, we wanted to test whether injury might also lead to greater susceptibility to peritoneal infection caused by a single common pathogen, Escherichia coli. Contrary to our expectation, we show herein that the LD50 for sham-injured mice was 103 CFU of E. coli, whereas the LD50 for burn-injured mice was 50 × 103 CFU at 7 days postinjury. This injury-associated enhanced resistance was apparent as early as 1 day after injury, and maximal resistance was observed at days 7 and 14. We found that burn-injured mice had higher numbers of circulating neutrophils and monocytes than did sham mice before infection and that injured mice were able to recruit greater numbers of neutrophils to the site of infection. Moreover, the peritoneal neutrophils in burn-injured mice were more highly activated than neutrophils from sham mice as determined by Mac-1 expression, superoxide generation, and bactericidal activity. Our findings suggest that the enhanced innate immune response that develops following injury, although it is commonly accepted as the mediator of the detrimental systemic inflammatory response syndrome, may also, in some cases, benefit the injured host by boosting innate immune antimicrobial defenses.

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“…The intracellular reaction between hydroethidine (HE, also known as dihydroethidium, DHE, Figure 1) and superoxide anion (O 2 ·-) results in the generation of a characteristic product, 2-hydroxyethidium cation (2-OH-E + , Figure 1) [1][2][3][4][5][6][7][8]. 1 For decades, this product was believed to be the ethidium cation (E + , Figure 1) [3][4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants

Zielonka¹,

Sarna²,

Roberts³

et al. 2006

Archives of Biochemistry and Biophysics

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Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here we report the direct measurement of the rate constant between hydroethidine and superoxide radical anion using the pulse radiolysis technique. This reaction rate constant was calculated to be ca. 2·10 6 M -1 s -1 in water:ethanol (1:1) mixture. The spectral characteristics of the intermediates indicated that the one-electron oxidation product of HE was different from the one-electron reduction product of ethidium (E + ). The HPLC-electrochemical measurements of incubation mixtures containing HE and the oxygenated Fenton's reagent (Fe 2+ /DTPA/H 2 O 2 ) in the presence of aliphatic alcohols or formate as a superoxide generating system revealed 2-OH-E + as a major product. Formation of 2-OH-E + by the Fenton's reagent without additives was shown to be superoxide dismutase-sensitive and we attribute the formation of superoxide radical anion to the one-electron reduction of oxygen by the DTPA-derived radical. Addition of tert-butanol, DMSO, and potassium bromide to the Fenton's system caused inhibition of 2-OH-E + formation. Results indicate that reducing and oxidizing radicals have differential effects on the formation of 2-OH-E + .

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Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes

Cited by 187 publications

References 30 publications

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations

Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations

Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function

Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants

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