Bead-based Fc-Receptor Phagocytosis Assay

PPD was biotinylated with sulfo-NHS-LC BIOTIN (ThermoFisher) and incubated with 1 μm fluorescent neutravidin beads (Invitrogen) at 4°C for 16 hours. Excess antigen was washed away. Antigen-coated beads were incubated with IgG samples (100 µg/mL) at 37°C for 2 hours to which THP1 cells (1 × 10⁵/well) were added and incubated further at 37°C for 16 hours. Bead uptake in fixed samples was measured on a BD LSRII. The integrated MFI (% bead-positive frequency × MFI/10 000) generated phagocytic scores [30 (link)].

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Lu L.L., Das J., Grace P.S., Fortune S.M., Restrepo B.I, & Alter G. (2020). Antibody Fc Glycosylation Discriminates Between Latent and Active Tuberculosis. The Journal of Infectious Diseases, 222(12), 2093-2102.

Publication 2020

sulfo-NHS-LC BIOTIN 1 μm fluorescent neutravidin beads BD LSRII

Antigen Cells Neutravidin Phagocytic Sulfo nhs lc biotin

Corresponding Organization : Ragon Institute of MGH, MIT and Harvard

Other organizations : The University of Texas Southwestern Medical Center, Harvard University, The University of Texas Health Science Center at Houston, The University of Texas Rio Grande Valley

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Variable analysis

independent variables

Concentration of IgG samples (100 µg/mL)

dependent variables

Bead uptake in fixed samples measured on a BD LSRII
Phagocytic scores (integrated MFI (% bead-positive frequency × MFI/10 000))

control variables

Incubation of antigen-coated beads with IgG samples at 37°C for 2 hours
Incubation of THP1 cells (1 × 10^5/well) with IgG-coated beads at 37°C for 16 hours
Biotinylation of PPD with sulfo-NHS-LC BIOTIN
Incubation of biotinylated PPD with 1 μm fluorescent neutravidin beads at 4°C for 16 hours
Washing away of excess antigen

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