Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2015/217258

Figure Lengend Snippet: 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 μ M), CRC (10 μ M), OLP (10 μ g/mL), QRC (10 μ M), GTE (12.5 μ g/mL), LYC (5 μ M), NAC (300 μ M), ALA (10 μ M), ALCAR (10 μ M), 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 μ M) 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. ∗∗ p ≤ 0.01 versus CTR-NGF; § p ≤ 0.05, §§ p ≤ 0.01 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 μ M), CRC (10 μ M), OLP (10 μ g/mL), QRC (10 μ M), GTE (12.5 μ g/mL), LYC (5 μ M), NAC (300 μ M), ALA (10 μ M), ALCAR (10 μ M), or CoQ (100 nM). Cells were loaded with DCFH-DA (10 μ M) 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 μ m. (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. ∗ p ≤ 0.05 versus No-NGF, ∗∗ p ≤ 0.01 versus CTR (ANOVA and Dunnett's multiple comparisons test).

Article Snippet: Cells were imaged at 40x magnification using a motorized Nikon Eclipse 90i (Nikon, Tokyo, Japan) fluorescence microscope equipped with a CCD camera (Hamamatsu-CoolSnap, Hamamatsu Corporation, Tokyo, Japan).

Techniques: Quantitation Assay, Fluorescence, Microscopy

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2015/217258

Figure Lengend Snippet: Analysis of mitochondrial function by MitoTracker Red/Green staining. (a) Representative merged images of neuronal PC12 cells maintained in the presence of NGF (CTR-NGF), NGF-deprived for 12 h (No-NGF), or exposed to NGF-free medium containing the antioxidants. NGF-differentiated PC12 cells were pretreated overnight with of RSV (10 μ M), QRC (10 μ M), OLP (10 μ g/mL), CRC (10 μ M), GTE (12.5 μ g/mL), LYC (5 μ M), NAC (300 μ M), ALA (10 μ M), ALCAR (10 μ M), CoQ (100 nM), or Sel (50 nM) before NGF withdrawal in the presence of the antioxidants. Cells were stained by MitoTracker Red and Green (50 and 200 nM, resp.) during the last 30 min of treatment and observed with a fluorescence microscope (Nikon) equipped with a CCD camera. Images were captured at 60x magnification. Arrowheads point to decreased ΔΨ m along neurites. Scale bar = 10 μ m. (b) MitoTracker Red fluorescence was quantified by NIH ImageJ, and the MFI was calculated by counting all cells (on average 200 cells) in about 20 random fields for each condition. Data, expressed as MFI, are the mean ± SEM of three experiments in duplicate. ∗ p ≤ 0.05 versus No-NGF, ∗∗ p ≤ 0.01 versus CTR (ANOVA and Dunnett's multiple comparisons test).

Article Snippet: Cells were imaged at 40x magnification using a motorized Nikon Eclipse 90i (Nikon, Tokyo, Japan) fluorescence microscope equipped with a CCD camera (Hamamatsu-CoolSnap, Hamamatsu Corporation, Tokyo, Japan).

Techniques: Staining, Fluorescence, Microscopy

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2015/217258

Figure Lengend Snippet: Effect of antioxidant cocktails on ROS levels and mitochondrial function following NGF deprivation. (a) Quantitation of ROS levels by FACS analysis of DCHF-DA fluorescence. Neuronal PC12 cells were preincubated overnight with Pool-1, Pool-2, or Pool-3 and then exposed to NGF-free medium for 6 hr. Cells were loaded with DCFH-DA (10 μ M) 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. ∗∗ p ≤ 0.01 versus CTR-NGF (ANOVA and Dunnett's multiple comparisons test). (b) Overlapping FACS profiles of a representative experiment. (c) Representative merged images of neuronal PC12 cells maintained in the presence of NGF (CTR) or NGF-deprived for 12 h. NGF-differentiated PC12 cells were pretreated overnight with Pool-1, Pool-2, or Pool-3 before NGF withdrawal in the presence of the indicated pools. Cells were stained by MitoTracker Red and Green (50 and 200 nM, resp.) during the last 30 min of treatment and observed with a fluorescence microscope (Nikon) equipped with a CCD camera. Images were captured at 60x magnification. Scale bar = 10 μ m. (d) MitoTracker Red fluorescence was quantified by NIH ImageJ, and the MFI was calculated by counting all cells (on average 200 cells) in about 20 random fields for each condition. Data, expressed as MFI, are the mean ± SEM of three experiments in duplicate. ∗ p ≤ 0.05 versus No-NGF, ∗∗ p ≤ 0.01 versus CTR (ANOVA and Dunnett's multiple comparisons test).

Article Snippet: Cells were imaged at 40x magnification using a motorized Nikon Eclipse 90i (Nikon, Tokyo, Japan) fluorescence microscope equipped with a CCD camera (Hamamatsu-CoolSnap, Hamamatsu Corporation, Tokyo, Japan).

Techniques: Quantitation Assay, Fluorescence, Staining, Microscopy