Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis

White-Owen, C; Alexander, J. Wesley; Sramkoski, R. Michael; Babcock, George F.

doi:10.1128/jcm.30.8.2071-2076.1992

Cited by 62 publications

(23 citation statements)

References 15 publications

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“…Similar experiences were also reported by White-Owen et al (25). The fluorescence intensity is known to fade when samples are exposed to light.…”

Section: Discussionsupporting

confidence: 87%

“…Phosphate buffered saline was added to all tubes to a final volume of 1 ml. Parafilm was placed over tubes, which were incubated in a shaking water bath at 37 C, with continuous agitation for 1,2,4,6,8,10,15,20,25,30,45, and 60 min, respectively. Samples were removed at each time period, centrifuged in a microcentrifuge for 1.5 min at 300 X g, and washed twice with ice-cold PBS to remove excess FITC bacteria.…”

Section: Methodsmentioning

confidence: 99%

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Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Heinzelmann

Gardner

Mercer-Jones

et al. 1999

Microbiology and Immunology

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Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

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“…Similar experiences were also reported by White-Owen et al (25). The fluorescence intensity is known to fade when samples are exposed to light.…”

Section: Discussionsupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Heinzelmann

Gardner

Mercer-Jones

et al. 1999

Microbiology and Immunology

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“…On the other hand, doubt as to whether reliable quantification of PR can be achieved still exists. A number of techniques have been proposed for the analysis of cellular phagocytic and killing processes (22,31,32), including flow cytometry (9,45), radiometric (37) and turbidimetric (19) assays, oxidative metabolism analysis (1 5 , 17), and chemiluminescence. Each of them possesses special characteristics and is to be preferred under different conditions: none has universally applicable features.…”

Section: Discussionmentioning

confidence: 99%

Quantitative analyses of plasma opsonizing activity and polymorphonuclear cell response during phagocytosis: standardisation of a chemiluminescent method

Farinelli

Setti

Puppo

et al. 1996

APMIS

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Although measurement of chemiluminescence has become a widespread tool in the study of phagocytosis of peripheral neutrophils, several problems linked to spontaneous fluctuation in chemiluminescence and the number of variables involved have occasionally either limited its usefulness for clinical and experimental purposes or compelled operators to take particular care when using the technique. In the present paper, sources of variability are investigated and most of the parameters involved are thoroughly analysed and step-by-step normalised. A stochastic calibration procedure for validation of the method is applied and a monofunctional test protocol for quantitative evaluation of plasma opsonizing activity in whole blood chemiluminescence is suggested. With regard to the goal of proposing a reverse monofunctional test, we discuss the reasons why further studies aimed at standardised evaluation of the cellular components are needed.

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“…PMN isolation PMN were isolated from EDTA anti-coagulatcd peripheral blood by centrifugation over neutrophil isolation media followed by hypotonic lysis of contaminating erythrocytes as previously described [10]. Briefly.…”

Section: Patient'imentioning

confidence: 99%

“…This parameter was used to compare, in rclati\'c terms, the receptor densities on the eel! populations of interest [10,13]. Non-PMN debris were excluded from analysis using forward and 90 angle light scattering.…”

Section: Flow Cytometrymentioning

confidence: 99%

Reduced PMN β2 integrins after trauma: a possible role for colony-stimulating factors

White-Owen

Hartmann²,

Alexander

et al. 1993

Clinical and Experimental Immunology

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SUMMARYThe /?i integrins are composed of a common 95-kD ^-subunit (CD 18) and one of three possible atsubunils: CDIIa. CDlIb. or CDMc. These molecules arc involved in neutrophil adhesion, diapodesis, chemotaxis and phagocytosis. In ihis study, the elTects oftrauniatic injury on neutrophi! expression of these a-subunits were investigated. Neutrophils from patients with severe trauma (rt = 30) were stained with fluorescent anti-CD 1 la, -CDI lb, or -CDl Ic. The percentage ofpositive neutrophils and the mean channel fluorescence were assayed by flow cytometry. In 10 patients and 10 normals, the elTects of granulocytc-colony stimulating factor (G-CSF) and granulocyte-niacrophage colony-stimulating factor (GM-CSF) on a-subunit expression were evaluated. Ninety-four ±2% (s.e.m.) of normal neutrophils were CDl la*, 89+1% were CDIIb' and 89 + 8% were CDllc+. Only 65 +2% of patient neutrophils wereCDlIa^,45±5% we reCDIlb* and 8+l%wereCDIlc'. Culture of normal neutrophils without colony-stimulating factors resulted in reduced expression of CDlla and CDlIc, bul up-regulation of CDllb. Down-regulalion of CDl la and CDIIc was partially reversed by colony-stimutating factors (30 U/ml). CD! lb receptor density was further upregulated by GM-CSF and G-CSF. Treatment of patient neutrophiis with colony-stimulating factors in culture resulted in up-regulation of a-subunits as well. GM-CSF appeared to have the greater effect. These results indicate that colony-stimulating factors may have a clinical role in improving ^2 integrin expression, and suggest a use in these infection-prone patients.

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Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis

Cited by 62 publications

References 15 publications

Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Quantitative analyses of plasma opsonizing activity and polymorphonuclear cell response during phagocytosis: standardisation of a chemiluminescent method

Reduced PMN β2 integrins after trauma: a possible role for colony-stimulating factors

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