Neutrophil Phagocytosis Assay with Immune Complexes
Phagocytosis score of primary human neutrophils was determined as described before (46 (link)). Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 50 μl of 1:200 diluted plasma. Primary cells were derived from ammonium-chloride-potassium (ACK) buffer-lysed whole blood from healthy donors and incubated with immune complexes for 1 h at 37°C. Neutrophils were stained for surface CD66b (Biolegend, CA, USA; clone G10F5) expression, fixed with 4% paraformaldehyde and analyzed by flow cytometry on a LSR Fortessa (BD).
Corresponding Organization : Ragon Institute of MGH, MIT and Harvard
Other organizations :
Massachusetts Institute of Technology, Brigham and Women's Hospital, Harvard University, HIV Netherlands Australia Thailand Research Collaboration, Chulalongkorn University, University Hospital of Geneva, University of Amsterdam
Biotinylated antigens coupled to fluorescent neutravidin beads
dependent variables
Phagocytosis score of primary human neutrophils
control variables
Plasma dilution (1:200)
Incubation time (1 h)
Incubation temperature (37°C)
Neutrophil source (healthy donors)
Neutrophil staining (CD66b expression)
Neutrophil fixation (4% paraformaldehyde)
Flow cytometry analysis (LSR Fortessa)
controls
Positive control: Not specified
Negative control: Not specified
Annotations
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