Neutrophil Apoptosis Assay

Isolated neutrophils (5×10⁶/mL) were cultured in 24 or 96-well plates in Iscove’s Modified Dulbecco’s Medium (IMDM) (in some experiments Roswell Park Memorial Medium (RPMI) was substituted) supplemented with 5% autologous serum (in some experiments 10% fetal calf serum (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 (GM-CSF; R&D Systems, Abingdon, UK) or E. coli LPS (Sigma, Dorset, UK) for 24 hours at 37°C, 5% CO₂. At stated 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 µg/mL propidium iodide (PI) before analysis on a BD FACS Scan, FACS Calibur or BD Accuri cell analyzer as described.16 (link) Cytocentrifuge preparations were stained with Diff-Quick (Gamidor, Didcot, UK) to assess for morphological changes of apoptosis.

Free full text: Click here

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

GM-CSF E. coli LPS Annexin V-FLUOS BD FACS Scan FACS Calibur BD Accuri

Annexin v Apoptosis At7519 Calcium chloride Cell E coli Fetal calf serum Fluos Glutamine Gm csf 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

Top 5 similar protocols

Variable analysis

independent variables

AT7519 (Astex Pharmaceuticals, Cambridge, UK)
Granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Abingdon, UK)
E. coli LPS (Sigma, Dorset, UK)

dependent variables

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

control variables

Isolated neutrophils (5×10^6/mL)
Iscove's Modified Dulbecco's Medium (IMDM) or Roswell Park Memorial Medium (RPMI) supplemented with 5% autologous serum, 10% fetal calf serum (FCS), or no serum
1% penicillin and streptomycin
1% L-glutamine
Incubation at 37°C, 5% CO2 for 24 hours
Resuspension of neutrophils in DPBS-/- supplemented with 25 mM calcium chloride for labeling with Annexin V-FLUOS and PI

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!