Water and H₂O₂ permeability depended on AQP3 expression in T cells. (a) The osmotic water permeability of CD4⁺ T cells isolated from WT and AQP3^−/− mice was measured based on the time course of scattered light intensity in response to a 150-mM inwardly directed mannitol gradient generated by stopped flow at 22°C. (left) Representative time course data showing responses to rapid changes in perfusate osmolality between 300 and 450 mOsm. (right) Averaged osmotic water permeability coefficients (P_f; SE; n = 5; *, P < 0.01). (b) Glycerol permeability was measured in response to a 150-mM inwardly glycerol gradient by stopped flow at 30°C (SE; n = 4). (c) H₂O₂ uptake into T cells. CD4⁺ T cells were incubated with 10–300 µM H₂O₂ for 15 s, and cellular H₂O₂ levels were detected using CM-H2DCFDA reagent by flow cytometric analysis. The mean fluorescence intensity (MFI) of CM-H2DCFDA fluorescence (SE; n = 4; *, P < 0.01, H₂O₂ added vs. control cells) is shown. (d) WT CD4⁺ cells were incubated with 5 µM DPI or 2,000 U/ml catalase for 30 min and followed with 500 ng/ml CXCL12 for 15–180 s at 37°C. CD4⁺ cellular H₂O₂ levels were detected using CM-H2DCFDA reagent by flow cytometry analysis (SE; n = 5; *, P < 0.01, CXCL12 treated vs. control cells). (e) Cellular H₂O₂ levels in WT and AQP3^−/− CD4⁺ cells after CXCL12 stimulation (500 ng/ml, 15 or 30 s; SE; n = 5; *, P < 0.01 vs. control cells). Each experiment was performed three times.