Intracellular H₂O₂ affects CXCL12-induced cell signaling and chemotaxis. (a–c) CD4⁺ T cells were incubated with 2,000 U/ml catalase (Cat), 5 µM DPI, or vehicle (Veh) for 30 min at 37°C and stimulated with 250 or 500 ng/ml CXCL12. (a) Quantification of Cdc42 active form (GTP bound) with G-LISA activation assay kit (SE; n = 4–5; *, P < 0.01). (b) Cytometric bead array–based quantification of phosphorylated Itk (SE; n = 4–5; *, P < 0.01). (c) Mean fluorescence intensity (MFI) of phalloidin–Alexa Fluor 660 in the CD4⁺ cells (SE; n = 5; *, P < 0.01). (d) Chemotaxis assay toward 100 ng/ml CXCL12 for 1 h (SE; n = 5; *, P < 0.01). (e and f) CD4⁺ T cells were incubated with 10–100 µM H₂O₂ for 1 min at 37°C. (e) Quantification of Cdc42 active form (GTP bound; SE; n = 4; *, P < 0.01). (f) The amount of phosphorylated Itk (SE; n = 4; *, P < 0.01). Experiments in a–d were performed in two other independent experiments and in e and f in one other experiment with similar results.