hpf staining kit  (GORYO Chemical)

Journal: Antioxidants

Article Title: Iron Chelation Reduces Intracellular Hydroxyl Radicals in Normal Human Dermal Fibroblasts Independently of Aging

doi: 10.3390/antiox14121437

Figure Lengend Snippet: Iron accumulation and increased ROS due to replicative senescence of NHDFs. ( a ) Intracellular free Fe 2+ fluorescence staining images and comparison of free Fe 2+ fluorescence intensity. Stained cells were observed in multiple fields of view, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young NHDFs (80 cells) and aged NHDFs (82 cells) obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. ( b ) Mitochondrial decline in aged NHDFs. Multiple fields of view of NHDF cells stained with Mito Tracker Deep Red were observed. Representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (81 cells) and aged (68 cells) NHDFs obtained from three independent experiments ( n = 3) were compared using box-and-whisker plots. ( c ) Increase in intracellular superoxide in aged NHDFs. Stained NHDFs were observed from multiple viewpoints, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (50 cells) and aged (73 cells) NHDFs obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. ( d ) Hydrogen peroxide release from NHDFs inhibited by NaN 3 . Hydrogen peroxide concentrations secreted into the culture medium over 24 h from young and aged NHDFs were measured with or without CAT inhibition. CAT was inhibited by NaN 3 (1.0 mM). Four independent measurements were performed ( n = 4), and the mean ± standard deviation is shown. ( e ) Intracellular HPF fluorescence staining image and comparison of HPF fluorescence intensity. Stained NHDFs were observed from multiple viewpoints, and representative images are shown. The scale bar indicates 100 μm. The fluorescence intensities of young (56 cells) and aged (56 cells) NHDFs obtained from three independent staining experiments ( n = 3) were compared using box-and-whisker plots. The boxes with quartiles shown in each section represent the median, 25th percentile, and 75th percentile. Whiskers indicate the maximum and minimum values within 1.5 times the interquartile range. Outliers are plotted separately. Dark boxes represent the young NHDFs, while light boxes represent the aged NHDFs. A Student’s t -test was performed, and a significance level of p < 0.05 was considered statistically significant. * indicates p < 0.05, while n.s indicates no significant difference. ( f ) The left panel shows a positive correlation between HPF fluorescence intensity in young (dark line and dots) and aged (pink line and dots) NHDFs and Fe(NO 3 ) 3 dose. The right panel shows the HPF fluorescence intensity in young (dark bars) and aged (pink bars) NHDFs as the mean and standard deviation for each Fe(NO 3 ) 3 dose. ANOVA and Tukey’s multiple comparison analysis were performed, with p < 0.05 indicating significant differences. * indicates p < 0.05. # indicates p < 0.05 vs. 0 μM. ♭ indicates p < 0.05 vs. 0 μM.

Article Snippet: To detect intracellular hydroxyl radicals, cells were stained with Hydroxyphenyl Fluorescein (HPF staining kit, Goryo Chemical., Sapporo, Japan) prepared at 50 μM in HBSS(+) for 30 min at 37 °C.

Techniques: Fluorescence, Staining, Comparison, Whisker Assay, Inhibition, Standard Deviation

Journal: Antioxidants

Article Title: Iron Chelation Reduces Intracellular Hydroxyl Radicals in Normal Human Dermal Fibroblasts Independently of Aging

doi: 10.3390/antiox14121437

Figure Lengend Snippet: Induction of hydroxyl radicals by hydrogen peroxide and its suppression by iron chelators. ( a ) HPF fluorescence intensity was measured independently three times in young NHDFs exposed to E-MEM supplemented with 0, 5, or 10 μM hydrogen peroxide for 24 h. Fluorescence intensity by hydrogen peroxide concentration is shown in box plots with quartiles. The number of cells measured for hydrogen peroxide concentrations of 0, 5, and 10 μM was 40 cells each. ( b ) Comparison of Ferro Orange fluorescence in young and aged NHDFs. Fluorescence intensity was measured in young and aged NHDFs from the control group, Fe(NO 3 ) 3 group, and Fe(NO 3 ) 3 + DFO group (114 cells per group) ( n = 5) and compared using a box plot. ( c ) Comparison of HPF fluorescence in young and aged NHDFs. Fluorescence intensity was measured in young and aged NHDFs from the control group, Fe(NO 3 ) 3 group, and Fe(NO 3 ) 3 + DFO group (56 cells per group) ( n = 3) and compared using a box plot. In each section, the dark boxes indicate the control group, the dotted boxes indicate the Fe(NO 3 ) 3 group, and the light boxes indicate the Fe(NO 3 ) 3 + DFO group. The boxes with quartiles shown in each section represent the median, 25th percentile, and 75th percentile. The whiskers indicate the upper and lower limits of the maximum values within 1.5 times the interquartile range. Outliers are plotted individually. One-way ANOVA and Tukey’s multiple comparison test were performed. A significance level of p < 0.05 was considered statistically significant. * indicates p < 0.05, and n.s indicates no significant difference.

Article Snippet: To detect intracellular hydroxyl radicals, cells were stained with Hydroxyphenyl Fluorescein (HPF staining kit, Goryo Chemical., Sapporo, Japan) prepared at 50 μM in HBSS(+) for 30 min at 37 °C.

Techniques: Fluorescence, Concentration Assay, Comparison, Control