Journal: bioRxiv

Article Title: IgA Potentiates NETosis in Response to Viral Infection

doi: 10.1101/2021.01.04.424830

Figure Lengend Snippet: (A) Primary human neutrophils were stimulated for 3 hours with antibody:IAV ICs generated from matched salivary IgA (sIgA) and serum IgA of healthy IAV-exposed donors (n=4). (B) Primary human neutrophils were incubated with an anti-CD89 (FcαRI) antibody prior to stimulation with IgG:IAV or IgA:IAV ICs (n=3) (C) Primary human neutrophils were stimulated with polyclonal IgA, polystyrene beads coated with Protein L, or polystyrene beads coated with protein L and IgA. For all experiments, NETosis was assessed by immunofluorescence microscopy analysis of cells co-stained for DNA (DAPI) and neutrophil elastase. NETosis in stimulated conditions was normalized to untreated cells (n=6). (D) Fluorescent polystyrene beads were coated with protein L, followed by either polyclonal IgG or IgA. Human neutrophils were isolated and incubated with the beads at a 500 beads/cell ratio. After washing, phagocytosis of beads was measured using a SpectraMax i3 plate reader (Molecular Devices) (n=3). (E) Purified Cal/09 was immobilized on glass coverslips prior to the addition of IgG or IgA. Primary human neutrophils were added to wells for 3 hours before being fixed and stained for quantification (n=3). Mean and SEM of independent experiments are shown. Statistical significance was evaluated by one-way ANOVA and Tukey post-hoc test. *, P < 0.05; **, P < 0.01.

Article Snippet: To block FcαRI, 20 μg/mL of mouse anti-human CD89 antibody (AbD Serotec) was added to neutrophils for 20 minutes at 4°C.

Techniques: Generated, Incubation, Immunofluorescence, Microscopy, Staining, Isolation, Purification