Phagocytosis score of primary human neutrophils was determined as described before(47 (link)). Biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher) and incubated with 10 μl 1:100 diluted plasma for 2h at 37°C to form immune-complexes. Primary neutrophils were derived from Ammonium-Chloride-Potassium (ACK) buffer lysed whole blood from healthy donors and incubated with washed immune complexes for 1h at 37°C. Afterwards, neutrophils were stained for surface CD66b (Biolegend, CA, USA; 1:100, clone: G10F5) expression, fixed with 4% para-formaldehyde and analyzed on a iQue analyzer (Intellicyt).
Protocol detail
Phagocytosis Assay for Primary Neutrophils
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Ammonium
Ammonium chloride
Antigens
Buffer
Cd66b
Chloride potassium
Clone
Donors blood
Formaldehyde
Human
Immune complexes
Neutravidin
Neutrophils
Phagocytosis
Plasma
Potassium
Corresponding Organization : Massachusetts General Hospital
Other organizations : Children's Clinical University Hospital, University College London
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Variable analysis
independent variables
- Biotinylated antigens coupled to FluoSphere NeutrAvidin beads
- Plasma dilution (1:100)
- Phagocytosis score of primary human neutrophils
- Incubation time of immune complexes (2h at 37°C)
- Incubation time of neutrophils with immune complexes (1h at 37°C)
- Neutrophil source (primary neutrophils derived from ACK-lysed whole blood of healthy donors)
- Neutrophil staining (CD66b expression)
- Neutrophil fixation (4% paraformaldehyde)
- No explicit positive or negative controls were mentioned in the provided information.
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