Figure 6. Live-cell Imaging of Peroxynitrite Production in Activated Macrophages.

(A) Mouse RAW264.7 cells were transfected with pnGFP-Ultra. Cells were next pretreated with IFN-γ (50 U/mL) and LPS (0.5 μg/mL) for 10 h. After LPS and IFN-γ were removed, cells were further treated with PMA (200 ng/mL) in HBSS for 1 h before imaging. Several control groups were used. In the “untreated” group were cells untreated with either of LPS, IFN-γ and PMA. In the “PMA” group were cells without pretreatment but directly treated with PMA for 1 h before imaging. In the “IFN-γ, LPS” group were cells pretreated with LPS and IFN-γ but without additional PMA treatment. In addition, cells in other control groups were co-treated with ONOO⁻ scavengers (urate or minocycline) or an iNOS inhibitor (1400W) when the PMA treatment was implemented. Representative bright field (left), fluorescence (middle) and overlay (right) images of each group were shown. Experiments were repeated three times with independent cultures and similar results were obtained. Scale bar, 20 μm.

(B) Quantification of fluorescence intensities of cells in each treatment group in panel A. Data are presented as mean ± 95% confidential intervals and black dots indicate fluorescent intensities of single cells (n = 15 individual cells from 3 independent replicates in each group). Statistical test was performed using Brown-Forsythe and Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test (***P < 0.001, **P < 0.01 and *P < 0.05).

(C) MTT cell viability assay of RAW264.7 cells in each treatment group. Raw data were normalized to pnGFP-Ultra-expressing cells untreated with either of LPS, IFN-γ and PMA. Data are presented as mean ± SD and black dots indicate the cell viability of individual replicates (n = 5 wells for each group). Statistical test was performed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (****P < 0.0001, ***P < 0.001, and **P < 0.01).

(D) Mouse RAW264.7 cells transiently expressing pnGFP-Ultra-Y.Cro (which contains a tyrosine-derived chromophore) were untreated or treated with LPS, IFN-γ, and PMA. Experiments were repeated three times with independent cultures and similar results were obtained. Scale bar, 20 μm.

(E) Quantification of fluorescence intensities of cells in each group in panel D. Data are presented as mean ± 95% confidential intervals and black dots indicate fluorescent intensities of single cells (n = 15 individual cells from 3 independent replicates in each group). Comparison was made using an unpaired two-tailed t-test (n.s., not significant, P > 0.05).