Mouse igg1 negative isotype control

10⁵ cells were added per well in 96-wells round bottom plates (Greiner bio-one, Cellstar) and stained with 10 µg/ml mouse anti-CD89 mAbs or a mouse IgG₁ negative isotype control (Biolegend). Subsequently, PE-labeled goat-anti-mouse IgG antibody (Jackson) was added. After final washings, cells were fixed in PBS/0.1% bovine serum albumin (BSA; Fitzgerald)/2%formaldehyde (37%; Sigma). Binding of anti-CD89 antibodies was determined with flow cytometry (FACS Cyan, Beckman Coulter), and expressed as signal-to-noise (S/N) ratios, ie, dividing measured geometric fluorescence intensity of anti-CD89 antibody by measured geometric fluorescence intensity of mouse IgG1 isotype control.

Transgenic mice expressing human CD89 and knock-in for human IgA were subcutaneously (sc) injected with 10 µl (7mg/ml) human-anti-mouse Collagen XVII IgA (auto)antibodies (Amsterdam UMC and Polpharma Biologics Utrecht) in the right ear and 10 µl PBS in the left ear as control. Injections were performed on day 0, 2, 4, 6, 8, 10 and 12. Mice were treated with 100 µl (1.5 mg/ml) of anti-human CD89 mouse antibody clone 10E7 (n=8, m/f) or a mouse IgG₁ negative isotype control (Biolegend) (n=4, m/f). Antibodies were injected intraperitoneally at day 7 and 11. Mice were monitored daily for discomfort. At the end of the experiment, day 14, mice were sacrificed, ears were excised and subsequently snap-frozen to use for cryosectioning and immunofluorescence staining.