Neutrophil Activation via ADNP Immune Complexes

For the ADNP assays^{40 (link)}, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating antigen coupled beads with 10 ul of 1:50 diluted plasma samples at 37 °C for 2 hours. Primary neutrophils were isolated as previously mentioned and 50,000 cells per well incubated with washed immune complexes at 37 °C for 1 hour. Following incubation, neutrophils were stained for surface CD66b (Biolegend, 1:100, clone: G10F5) expression and fixed with 4% para-formaldehyde. Analysis was done on an iQue analyzer (Intellicyt).

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Bartsch Y.C., Chen J.W., Kang J., Burns M.D., St Denis K.J., Sheehan M.L., Davis J.P., Edlow A.G., Balazs A.B., Yonker L.M, & Alter G. (2022). BNT162b2 induces robust cross-variant SARS-CoV-2 immunity in children. NPJ Vaccines, 7, 158.

Publication 2022

FluoSphere NeutrAvidin beads iQue analyzer

Antigen Cd66b Cells Clone Formaldehyde Immune complexes Neutravidin Neutrophils Plasma

Corresponding Organization :

Other organizations : Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Reproductive Science Center

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Variable analysis

independent variables

Antigen coupled beads
Plasma samples (1:50 dilution)

dependent variables

Neutrophil CD66b expression

control variables

Neutrophil isolation method
Incubation conditions (37 °C, 1 hour)
Staining and fixation protocol

controls

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

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