Oxidative Medicine and Cellular Longevity / 2015 / Article / Fig 4

Research Article

Neuroprotection by Cocktails of Dietary Antioxidants under Conditions of Nerve Growth Factor Deprivation

Figure 4

Effect of antioxidants on ROS levels following NGF deprivation. (a) Quantitation of ROS levels by FACS analysis of DCHF-DA fluorescence. Neuronal PC12 cells were preincubated overnight with RSV (10 μM), CRC (10 μM), OLP (10 μg/mL), QRC (10 μM), GTE (12.5 μg/mL), LYC (5 μM), NAC (300 μM), ALA (10 μM), ALCAR (10 μM), CoQ (100 nM), or Sel (50 nM) and then changed for 6 h to NGF-free medium containing the same antioxidant. Cells were loaded with DCFH-DA (10 μM) during the last 30 min of treatments and flow cytometric measurements (Geo-mean values) were taken on 10,000 cells contained in the gated regions. Data are the mean ± SEM of three experiments in duplicate. versus CTR-NGF; , versus No-NGF (ANOVA and Dunnett’s multiple comparisons test). (b) FACS profiles of a representative experiment. (c) Representative images of fluorescence microscopy analysis of ROS levels on neuronal PC12 cells after NGF deprivation for 6 h in the presence of RSV (10 μM), CRC (10 μM), OLP (10 μg/mL), QRC (10 μM), GTE (12.5 μg/mL), LYC (5 μM), NAC (300 μM), ALA (10 μM), ALCAR (10 μM), or CoQ (100 nM). Cells were loaded with DCFH-DA (10 μM) during the last 30 min of treatments and observed under a fluorescence microscope (Nikon) equipped with a CCD camera. Images were captured at 40x magnification. Scale bar = 10 μm. (d) Quantitation of DCHF-DA positive cells by NIH ImageJ. MFI was calculated on about 150 cells in 10 random fields for each condition. Data are the mean ± SEM of three experiments in duplicate. versus No-NGF, versus CTR (ANOVA and Dunnett’s multiple comparisons test).
(a)
(b)
(c)
(d)