Neutrophil Apoptosis Assay Protocol

Isolated neutrophils (5×10⁶/ml) were cultured in 24 or 96 well plates in IMDM (in some experiments RPMI was substituted) supplemented with 5% autologus serum (in some experiments 10% FCS or no serum was substituted), 1% penicillin and streptomycin and 1% L-glutamine alone or in the presence of AT7519 (Astex Pharmaceuticals, Cambridge, UK), Granulocyte Macrophage-Colony Stimulating Factor (R&D Abingdon, UK) or E. coli Lipopolysacharide (LPS), (Sigma, Dorset, UK). for 24 hours at 37°C, 5% CO₂. At given time points neutrophils were resuspended (1:5) in DPBS^-/- supplemented with 25 mM calcium chloride and labelled with Annexin V-FLUOS (Sigma) at 1:500 and 1.0 mg/ml propidium iodide (1:300) before analysis on a BD FACS Scan, FACS Calibur, or BD Accuri cell analysers as described(19 (link)). Cytocentrifuge preparations were stained with Diff-Quick (Gamidor, Didcot, UK), to assess for morphological changes of apoptosis.

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Gray R.D., Hardisty G., Regan K.H., Smith M., Robb C.T., Duffin R., Mackellar A., Felton J.M., Paemka L., McCullagh B.N., Lucas C.D., Dorward D.A., McKone E.F., Cooke G., Donnelly S.C., Singh P.K., Stoltz D.A., Haslett C., McCray P.B., Whyte M.K., Rossi A.G, & Davidson D.J. (2017). Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis. Thorax, 73(2), 134-144.

Publication 2017

Granulocyte Macrophage-Colony Stimulating Factor Annexin V-FLUOS BD FACS Scan FACS Calibur BD Accuri

Annexin v Apoptosis At7519 Calcium chloride Cell E coli Fluos Glutamine Granulocyte macrophage colony stimulating factor Neutrophils Penicillin Pharmaceuticals Propidium iodide Scan Serum Streptomycin

Corresponding Organization : University of Edinburgh

Other organizations : University of Iowa, St. Vincent's University Hospital, Tallaght University Hospital, Trinity College Dublin, University of Washington Medical Center

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Variable analysis

independent variables

AT7519 (Astex Pharmaceuticals, Cambridge, UK)
Granulocyte Macrophage-Colony Stimulating Factor (R&D Abingdon, UK)
E. coli Lipopolysacharide (LPS) (Sigma, Dorset, UK)

dependent variables

Neutrophil apoptosis (measured by Annexin V-FLUOS and propidium iodide staining and flow cytometry)
Neutrophil morphological changes (assessed by Diff-Quick staining and cytocentrifuge preparations)

control variables

Isolated neutrophils (5×10^6/ml)
Culture in IMDM or RPMI supplemented with 5% autologous serum, 10% FCS, or no serum
1% penicillin and streptomycin
1% L-glutamine
Culture for 24 hours at 37°C, 5% CO2

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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