Journal: Biomedicines

Article Title: Live Imaging of Nitric Oxide Dynamics Reveals Cell Type-Specific NO Signaling in Air–Liquid Interface Cultures of Human Sinonasal Epithelial Cells

doi: 10.3390/biomedicines14061340

Figure Lengend Snippet: Nitric oxide production in human sinonasal epithelial cells in ALI culture using NO-specific fluorescent indicators DAF-FM DA. Representative LSCM fluorescence images of ALI cultures after 4 weeks of differentiation. ( a ) Cells show distinct NO-related fluorescence as detected by DAF-FM fluorophores (green). ( b ) AlexaFluor 555-labeled staining of ac-tubulin (red) shows abundant apical cilia with a cobblestone epithelial pattern. ( c ) Nuclear DAPI stain is shown in blue. Scale bars: 15 μm.

Article Snippet: ALI cultures were incubated on the apical side with 10 μM DAF-FM diacetate (#SK1004-01; Goryo Chemical, Sapporo, Japan) in HBSS for 30 min. DAF-FM has optimal excitation and emission wavelengths of 495 and 515 nm, respectively.

Techniques: Fluorescence, Labeling, Staining

Journal: Biomedicines

Article Title: Live Imaging of Nitric Oxide Dynamics Reveals Cell Type-Specific NO Signaling in Air–Liquid Interface Cultures of Human Sinonasal Epithelial Cells

doi: 10.3390/biomedicines14061340

Figure Lengend Snippet: Comparison of nitric oxide (NO)-related fluorescence signals between ciliated and non-ciliated sinonasal epithelial cells in 4-week ALI cultures as revealed by DAF-FM fluorescence. Each data point represents the mean fluorescence intensity measured within a single region of interest (ROI; 1924 pixels) corresponding to an individual cell. ROIs were obtained from ALI cultures derived from independent donors. Bar graphs show mean ± SEM. ( a ) Administration of 0.5 mM L-arginine increased DAF-FM fluorescence intensity, with ciliated cells exhibiting significantly higher fluorescence than non-ciliated cells. ( b ) The non-selective NOS inhibitor L-NAME (0.5 mM) markedly reduced fluorescence signals in both cell types. ( c ) The selective iNOS inhibitor 1400W (100 μM) reduced fluorescence intensity but preserved significantly higher fluorescence in ciliated cells. Statistical comparisons between ciliated and non-ciliated cells were performed using donor-level mean fluorescence values ( n = 5 donors) and analyzed using a paired t -test. * p < 0.05; N.S., not significant.

Article Snippet: ALI cultures were incubated on the apical side with 10 μM DAF-FM diacetate (#SK1004-01; Goryo Chemical, Sapporo, Japan) in HBSS for 30 min. DAF-FM has optimal excitation and emission wavelengths of 495 and 515 nm, respectively.

Techniques: Comparison, Fluorescence, Derivative Assay