Flow Cytometry of Cell Surface Markers

Flow cytometry was performed as described previously [41 (link)] with some modifications. A human Fc receptor-binding inhibitor polyclonal antibody (50-112-9053, Fisher Scientific) was used as a blocking agent prior to adding the primary antibodies in all B cell surface-labeling experiments. Rabbit anti-human prostasin sera or pre-immune rabbit sera [7 (link)] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution. A goat anti-rabbit IgG-cyanine-Cy™3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was used as the secondary antibody at 1:200 dilution. The labeled cells (10,000) were analyzed using a CytoFLEX S flow cytometer with the laser configuration of V2B2Y3R2, operated by CytExpert software v2.3 (Beckman Coulter, Brea, CA, USA). The data were analyzed with FlowJo™ software v10.8.1.