Computational protocol: Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

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Protocol publication

[…] NIH-3T3 fibroblasts were grown to confluent in 12-well plates in DMEM containing 10% FBS. Then fibroblasts were deprived in DMEM with 0.1% FBS for 6–8 hours. MSC were grown to confluent in 12-well plates in ASBM containing 10% AdvanceStem Cell Growth Supplement. Then MSC were deprived in ASBM without Supplement for 24 hours. The cell monolayers were scratched with a 1-ml pipette tip 30 min before stimulation with PDGF-BB (final 10 ng/ml) or EGF (final 20 ng/ml), briefly rinsed with the medium and supplied with DMEM containing 0.1% FBS. The inhibitors or vehicle were added 30 min prior to stimulation to final concentrations of 10 μM for LY294002 (PI3-kinase inhibitor), 10 μM for U0126 (Erk1/2 activation inhibitor), 2 mM for apocynin (Nox inhibitor) and 40 U/ml for PEG-conjugated (membrane permeable) catalase. DPI and ebselen were added to 5 μM and 20 μM, respectively, 5 min prior to stimulation to minimize their cytotoxic effects. Then culture plates were transferred on to the microscopic stage of motorized Nikon Ti inverted microscope (Nikon, Japan) equipped with the 4x objective, on-stage culture box, temperature controller set to 37°C and continuous carbogen administration unit. The time-lapse series were continuously acquired every 10 min over 24 h using cooled CCD camera (Nikon, Japan) and the 'Mark and Find' application in NIS Elements (Nikon, Japan) to achieve simultaneous image acquisition in all 12 wells of the plate. This frequency ensures that in each series two successive displacements of a cell are resolved and all cell divisions are captured to be excluded from the analysis later on. The time series were analyzed by manual tracking all cells on the edge of the experimental wounds and measuring their velocity in 2 randomly chosen positions of the wounded areas using free ImageJ software. Routinelly, 50–60 cells were tracked for each data point in one of six independent experiments, accounting for more than 300 cells per point in total. [...] Statistical analysis was performed using SigmaPlot version 12.5 (Systat Software Inc). All experiments were performed at least three times. Results are shown as mean ± Standard Error of Mean (SEM). The Shapiro-Wilk test was used to test normal distribution. Statistical analysis of two groups was done using Student’s t-test. Multiple comparisons were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis test (one-way ANOVA on ranks, for not normally distributed data). Post hoc analysis of pairwise comparisons after multiple testing was achieved with Tukey or Dunnett’s tests for one-way ANOVA and with Dunn’s test for ANOVA on ranks. The p values < 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Chemicals Hydrogen Peroxide