Neutrophil Phagocytosis Assay with IgA-Depleted Plasma

Neutrophil phagocytic activity of IgA depleted plasma samples was tested on primary human neutrophils^{44 (link)}. Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated 1:100 diluted plasma. Primary neutrophils were incubated with immune complexes for 1h at 37°C. Next, cells were stained for surface CD66b expression with a 1:100 diluted antibody (Biolegend, CA, USA; clone: G10F5), fixed with 4% paraformaldehyde, analyzed by flow cytometry, and phagocytosis score calculated as described above (Supplemental Figure 14).

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Bartsch Y.C., Wang C., Zohar T., Fischinger S., Atyeo C., Burke J., Kang J., Edlow A.G., Fasano A., Baden L.R., Nilles E.J., Woolley A.E., Karlson E.W., Hopke A.R., Irimia D., Fischer E.S., Ryan E.T., Charles R., Julg B.D., Lauffenburger D.A., Yonker L.M, & Alter G. (2021). Humoral signatures of protective and pathological SARS-CoV-2 infection in children. Nature medicine, 27(3), 454-462.

Publication 2021

fluorescent neutravidin beads

Antibody Antigens Cd66b Cells Clone Flow cytometry Human Immune complexes Neutravidin Neutrophils Paraformaldehyde Phagocytic Phagocytosis Plasma

Corresponding Organization :

Other organizations : Ragon Institute of MGH, MIT and Harvard, Massachusetts Institute of Technology, Massachusetts General Hospital, Brigham and Women's Hospital, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Biotinylated antigens coupled to fluorescent neutravidin beads

dependent variables

Neutrophil phagocytic activity
Surface CD66b expression

control variables

1:100 diluted plasma samples
Primary human neutrophils
Incubation time of 1 hour at 37°C
4% paraformaldehyde fixation
Flow cytometry analysis

controls

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

Annotations

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