Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers

Eggleton, Paul; Gargan, R.A.; Fisher, Derek

doi:10.1016/0022-1759(89)90425-0

Cited by 106 publications

(55 citation statements)

References 14 publications

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“…Human neutrophils were prepared as described previously (16,18). Briefly, 10 ml of heparinized blood was obtained from healthy donors and mixed 1:1 with PBS containing 3% dextran T500.…”

Section: Methodsmentioning

confidence: 99%

α-Enolase of Streptococcus pneumoniae Induces Formation of Neutrophil Extracellular Traps

Mori

Yamaguchi

Terao

et al. 2012

Journal of Biological Chemistry

102

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“…Human neutrophils were prepared as described previously (16,18). Briefly, 10 ml of heparinized blood was obtained from healthy donors and mixed 1:1 with PBS containing 3% dextran T500.…”

Section: Methodsmentioning

confidence: 99%

α-Enolase of Streptococcus pneumoniae Induces Formation of Neutrophil Extracellular Traps

Mori

Yamaguchi

Terao

et al. 2012

Journal of Biological Chemistry

102

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“…Control, blocking antibodies (10 g/ml), or SK inhibitor SKi (1 mol/L, Cayman Chemical Co.) were added for 30 minutes before cell perfusion. Using published methods, heparinized whole blood was diluted 1:10 in Hank's balanced salt solution (Sigma) 27 or used to isolate neutrophils 28 and cells (diluted blood or 1 ϫ 10 6 isolated neutrophils) were perfused across the substratum by a syringe pump (NE-1000, New Era Pump System, Inc, Wartagh, NY) at a constant rate of 2 dynes/cm 2 for 5 minutes followed by Hank's balanced salt solution wash. Interactions of unlabeled cells were visualized using ϫ10/0.3 NA objectives and phase-contrast microscopy on an inverted microscope and images were recorded using a digital camera (Olympus IX70 and SIS F-view, Olympus). Five random areas per dish were recorded for analysis using AnalySIS Life Sciences software (Olympus).…”

Section: Parallel Plate Flow Chamber Assaymentioning

confidence: 99%

Tumor Necrosis Factor-Induced Neutrophil Adhesion Occurs Via Sphingosine Kinase-1-Dependent Activation of Endothelial α5β1 Integrin

Sun

Pitson

Bonder

2010

The American Journal of Pathology

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Leukocyte recruitment plays a major role in the immune response to infectious pathogens, as well as during inflammatory and autoimmune disorders. The process of leukocyte extravasation from the blood requires a complex cascade of adhesive events between the leukocytes and the endothelium, including initial leukocyte rolling, adhesion, and finally transendothelial migration. Current research in this area aims to identify the key leukocyte subsets that initiate a given disease and to identify the trafficking molecule(s) that will most specifically inhibit those cells. Herein we demonstrate that tumor necrosis factor (TNF)␣ activates the integrin ␣ 5 ␤ 1 without altering total expression levels of ␤ 1 integrin on human umbilical vein endothelial cells. Moreover, our studies suggest that TNF␣-induced ␤ 1 activation is dependent on sphingosine kinase-1, but independent of the sphingosine-1-phosphate family of G protein-coupled receptors. We also show, using a parallel plate flow chamber assay, that neutrophil adhesion to TNF␣-activated endothelium can be attenuated by blocking ␣ 5 ␤ 1 or its ligand angiopoietin-2. These observations add new complexities that broaden the accepted concept of cellular trafficking with neutrophil adhesion to TNF␣ activated endothelial cells being sphingosine kinase-1, ␣ 5 ␤ 1 , and angiopoietin-2 dependent. Moreover, this work supports the notion that sphingosine kinase-1 may be the single target required for an effective broad spectrum approach to combat inflammation and immune disorders.

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“…11 A fluorescent method was employed to test cell viability. 12 Cytospin preparation (Shandon Cyto-2) was performed at 10 g on low acceleration for five minutes.…”

Section: Immunofluorescencementioning

confidence: 99%

ELISA is the superior method for detecting antineutrophil cytoplasmic antibodies in the diagnosis of systemic necrotising vasculitis.

Harris¹,

Chang²,

Vadas³

et al. 1999

J Clin Pathol

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Background-Antineutrophilcytoplasmic antibodies (ANCA) have been used as a diagnostic marker for systemic necrotising vasculitis, a disease classification which includes Wegener granulomatosis, microscopic and classic polyarteritis nodosa, and Churg Strauss disease. Objective-To compare the diagnostic value of the two methods for detecting these antibodies-immunofluorescence and enzyme linked immunosorbent assay (ELISA)-with respect to biopsy proven active systemic necrotising vasculitis in a clinically relevant population. Methods-A prospective study to ascertain the patient's diagnosis at the time of each of the 466 requests for ANCA received at one laboratory over a nine month period, and allocate each to one of five diagnostic groups: active and inactive biopsy proven systemic necrotising vasculitis, suspected systemic necrotising vasculitis, low probability systemic necrotising vasculitis, and not systemic necrotising vasculitis. Results-ELISA was superior to immunofluorescence in the diagnosis of systemic necrotising vasculitis because it was less likely to detect other diseases. This was reflected in its specificity of 97% and positive predictive value of 73%, compared with 90% and only 50% for immunofluorescence (p = 0.0006 and p = 0.013, respectively). ELISA had a negative predictive value of 98% which was not significantly diVerent to immunofluorescence. ELISA was technically superior. Conclusions-ELISA is the superior method of ANCA detection in the diagnosis of systemic necrotising vasculitis and should be used in conjunction with a compatible clinical picture and histological evidence. (J Clin Pathol 1999;52:670-676)

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Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers

Cited by 106 publications

References 14 publications

α-Enolase of Streptococcus pneumoniae Induces Formation of Neutrophil Extracellular Traps

α-Enolase of Streptococcus pneumoniae Induces Formation of Neutrophil Extracellular Traps

Tumor Necrosis Factor-Induced Neutrophil Adhesion Occurs Via Sphingosine Kinase-1-Dependent Activation of Endothelial α5β1 Integrin

ELISA is the superior method for detecting antineutrophil cytoplasmic antibodies in the diagnosis of systemic necrotising vasculitis.

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