Fitc gr 1

2 µm Fluoresbrite^® yellow/green carboxylate microspheres (Polysciences, 098475) were coated with BSA by incubation with 1 mg/ml BSA in 50 mM MES buffer (pH 6.7) containing 20 mg/ml EDAC (Sigma, E7750) for 1 h at RT, washed, and stored in 10 mM Tris pH 8, 0.05% BSA. To opsonise the beads with IgG, they were washed in PBS, incubated with anti-BSA IgG (1:250 in PBS) for 1 h at RT, washed, and resuspended in HBSS⁺⁺⁺⁺. Alternatively, beads remained unopsonised. Purified neutrophils were resuspended at 1x10⁷/ml in HBSS⁺⁺⁺⁺ and primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min at 37°C as described above. 100 μl of cells were combined with 100 μl BSA-coated or IgG-opsonised beads at 1 x 10⁸/ml (10 particles/neutrophil) in a 24-well plate containing sterile glass coverslips, and were incubated for 15 min at 37˚C. Cells were fixed with 4% PFA in for 15 min and stained with FITC-Gr1 (BD Pharmingen, 553126, 1:800) containing 1% Fc block and mounted in ProLong Gold Antifade mountant (Life Technologies). Image analysis was done with Fiji, using the FITC-Gr1 signal at the phagosomal membrane to distinguish phagosomes from particles attached to the outside of the cell. The number of particles taken up per cell, and the percentage of neutrophils containing at least one particle were enumerated manually.