ADNP Immune Complex Phagocytosis Assay

The ADNP assay was adapted from Karsten et al.^{83 (link)}. Antigens were coupled to beads and immune complexes were formed as described for ADCP. Neutrophils were isolated from fresh whole Acid Citrate Dextrose (ACD) blood using EasySep Direct Human Neutrophil Isolation kit (Stem Cell,#19666), resuspended in R10, and added to plates at a concentration of 5 × 10⁴ cells/well. The plates were incubated for 30 min at 37 ˚C. The neutrophil marker CD66b (Pacific Blue conjugated anti-CD66b; BioLegend, #305112) was used to stain cells (1:100 dilution). Cells were fixed for 10 min in 4% PFA. Fluorescence was acquired with a Stratedigm 1300EXi cytometer and phagocytic score was calculated as described for ADCP.

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Roy V., Jung W., Linde C., Coates E., Ledgerwood J., Costner P., Yamshchikov G., Streeck H., Juelg B., Lauffenburger D.A, & Alter G. (2023). Differences in HPV-specific antibody Fc-effector functions following Gardasil® and Cervarix® vaccination. NPJ Vaccines, 8, 39.

Publication 2023

EasySep Direct Human Neutrophil Isolation kit Pacific Blue conjugated anti-CD66b 1300EXi cytometer

Acid citrate dextrose Antigens Assay Blood Cd66b Cells Dilution Fluorescence Human Immune complexes Isolation Neutrophil Phagocytic Stain Stem cell

Corresponding Organization :

Other organizations : Ragon Institute of MGH, MIT and Harvard, National Institute of Allergy and Infectious Diseases, National Institutes of Health, University Hospital Bonn, Massachusetts Institute of Technology

Top 5 similar protocols

Variable analysis

independent variables

Antigens coupled to beads

dependent variables

Phagocytic score

control variables

Neutrophils isolated from fresh whole Acid Citrate Dextrose (ACD) blood using EasySep Direct Human Neutrophil Isolation kit
Neutrophils resuspended in R10
Neutrophils added to plates at a concentration of 5 × 10^4 cells/well
Plates incubated for 30 min at 37 ˚C
Neutrophil marker CD66b (Pacific Blue conjugated anti-CD66b) used to stain cells (1:100 dilution)
Cells fixed for 10 min in 4% PFA

controls

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

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