Zymosan Induced peritonitis and flow cytometry:
C57BL/6J, CBA/CaCrl (WT) or CBA/CaHN-Btk xid/J
(XID) mice were administered 100 µg zymosan A (Sigma-Aldrich) in PBS or
vehicle intraperitoneally. After 2, 4, 16 or 48 h mice were sacrificed
and peritoneal exudates collected by lavage with 7 ml of ice-cold
sterile PBS with 5 mM EDTA. Total cell counts and cellular composition
of peritoneal exudate were determined as previously described (Cash et
al., 2009). Antibodies were used to identify neutrophils
(CD11b+LyGC+Ly6G+),
monocytes
(CD11b+LyGC+Ly6G-)
and B-cells (CD11b-BV220+) were
purchased from Biolegend (San Diego, CA). Data was acquired using a BD
LSRFortessa cytometer, and data analysed using FlowJo. Dosing regimen
was determined from assessment of previously published doses of
ibrutinib known to inhibit BTK in vivo (O’Riordan et al., 2019b),
pre-dosing strategy was used as a proof of principle methodology for
drug efficacy. Delivery route (oral gavage) has been previously
demonstrated.