Soluble uric acid affects β₂ integrin internalization and recycling in human neutrophils. (A) Schematic illustrating the process of integrin internalization and recycling in neutrophils (detailed information is described in supplemental Methods). (B) Human neutrophils were isolated from healthy individuals and preincubated with or without 10 mg/dL sUA for 30 minutes prior to stimulation with CXCL8. The integrin internalization assay for anti-human CD18 in CXCL8-activated neutrophils was performed using flow cytometry. The percentage (%) of internalized CD18 in CXCL8-activated neutrophils was determined after 10 and 30 minutes (n = 7 per group). (C) Immunoblotting for CD18 and β-actin was performed, and the protein CD18/β-actin ratio to medium quantified (n = 4 per group). (D) The integrin internalization assay was performed and the percentage of internalized MAC-1 after 10 and 30 minutes determined by flow cytometry (n = 4 per group). (E) The integrin recycling assay was performed and the percentage of recycled CD18 after 10 and 30 minutes determined by flow cytometry (n = 4 per group). (F-H) Cytoskeletal dynamics were determined in CXCL8-activated neutrophils (n = 4 per group) via flow cytometry using the forward scatter (FSC-H) for quantifying cellular size (F-G) and the dye DiBAC₄(3) for quantification of membrane potential (as MFI) (H). (I-J) Intracellular pH was determined as MFI of the dye SNARF-1 (MFI) (I) by flow cytometry and the intracellular pH calculated (J) (n = 4 per group). Data are mean plus or minus SD. *P < .05, **P < .01. Ctrl, control; ns, not significant by 2-way ANOVA.