Computational protocol: A synthetic cell permeable antioxidant protects neurons against acute oxidative stress

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Protocol publication

[…] SH-SY5Y cells were seeded at 1.25 × 105 cells/cm2 on coverslips and incubated for 24 hours prior to addition of Proxison or vehicle (DMSO) for 30 minutes. Then 400 μM tBHP was added for 4.5 hours when cells were fixed with 4% PFA for 20 minutes and washed three times with PBS. Immunocytochemistry was performed by the addition of blocking buffer (0.1% Triton X-100 or 0.3% Triton-X100 for nuclear staining, 2% goat serum, PBS) for 30 minutes. Primary antibodies rabbit IgG NRF2 (1:800, C-20, Santa Cruz), mouse IgG2b 8-OHdG (1:1000, 15A3, abcam) in blocking buffer were added to cells for 16 hours at 4 °C, when they were washed 3 times in PBS with 0.1% Triton X-100. Secondary antibodies (Life Technologies) in blocking buffer were incubated with cells for 2 hours at room temperature in the dark, when they were washed 3 times in PBS with 0.1% Triton X-100. DAPI (10–50 μg/ml, Life Technologies) in PBS was added for 10 minutes. Cells were imaged using Leica TCS SPE confocal microscope. NRF2 images were quantified using ImageJ. The z-stack images were flattened and a mask was created using the DAPI channel. This mask was then used to calculate the nuclear red fluorescence intensity. The mean fluorescence from each image (2–4 images) and from each coverslip was calculated. The 8-OHdG images were quantified using ImageJ. The z-stack images were flattened and a mask was created using the DAPI channel, this mask included the nuclei and the area surrounding the nuclei by reducing the threshold. This mask was then used to calculate the nuclear green fluorescence intensity. The mean fluorescence from each image was calculated with ImageJ. [...] Statistical Analysis was performed in Minitab using the Analysis of Variance (ANOVA) statistical test with a Tukey’s post-hoc test. Raw data used to generate all graphs can be found at LabArchives DOI 10.6070/H44X55WM. […]

Pipeline specifications

Software tools ImageJ, LabArchives
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio
Diseases Parkinson Disease, Neurodegenerative Diseases, Stroke
Chemicals Flavonoids, Oxygen