Fig. 3

Effects of tirabrutinib on MPO and anti-MPO-IC-induced NET formation. Human peripheral blood neutrophils were allowed to react with tirabrutinib at concentrations of 0, 2.5, 5.0, and 10 nM for 1 h at 37 °C. Neutrophils were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 10⁵/well) and allowed to settle at 37 °C. Four hours later, the samples were fixed with 4% PFA for 15 min at RT and mounted with a DAPI-containing mounting solution (a). Arrowheads indicate NETs. Human peripheral blood neutrophils were allowed to react with tirabrutinib at concentrations of 0, 2.5, 5.0, and 10 nM for 1 h at 37 °C, followed by 5 ng/ml TNF-α for 15 min at 37 °C. Neutrophils were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 10⁵/well) and allowed to settle at 37 °C. Four hours later, cells were collected, resuspended in ethylenediaminetetraacetic acid-PBS, and analyzed by FCM to examine forward scatter (FSC) and side scatter (SSC) profiles (b). This analysis focused on swollen neutrophils [R8 gate (whole neutrophils)-R7 gate (normal size neutrophils)]. The value of samples without stimulation was set as 1. *p < 0.05; **p < 0.01