Journal: International Journal of Molecular Sciences
Article Title: High-Contrast Stimulation Potentiates the Neurotrophic Properties of Müller Cells and Suppresses Their Pro-Inflammatory Phenotype
doi: 10.3390/ijms23158615
Figure Lengend Snippet: The combination of high-contrast stimulation and BDNF treatment augmented the neurodifferentiation potential and suppressed the pro-inflammatory phenotype of MCs. Quantification of ( a ) live MCs (fixable viability dye negative cells), ( b ) BrdU incorporation by MCs, ( c ) SOX2 + /nestin + MCs, and ( d ) SOX2 + /GFAP + MCs that were either stimulated by high contrast or unstimulated and cultured in the absence or presence of BDNF at the indicated concentrations (0.1 nM, 1 nM, 10 nM). Densitometric Western blot analysis of p-p65 expression in the ( e ) nuclear and ( f ) cytoplasmic extracts of high-contrast-stimulated and unstimulated MCs that were cultured in the absence or presence of BDNF at the indicated concentrations (0.1 nM, 1 nM, 10 nM). ( g ) Representative Western blot images of p-p65 expression in the nuclear and cytoplasmic extracts of high-contrast-stimulated and unstimulated MCs with or without BDNF treatment. Each experiment was repeated three times. The data are the means ± SEM. The results are presented as fold changes from the control group (unstimulated MCs without BDNF treatment). Differences between the stimulated and unstimulated groups treated with the same concentration of BDNF: + p < 0.05, ++ p < 0.01, +++ p < 0.001. Differences between the high-contrast-stimulated group treated with BDNF and the high-contrast-stimulated group without BDNF treatment: ** p < 0.01, *** p < 0.001. Differences between the unstimulated group treated with BDNF and the unstimulated group without BDNF treatment: ## p < 0.01, ### p < 0.001 (two-way ANOVA followed by Tukey’s post-hoc test).
Article Snippet: Next, cells were fixed using Fixation/Permeabilization Diluent (00-5223-56; Thermo Fisher Scientific) and intracellularly stained with anti-BrdU FITC-conjugated antibody (11-5071-42; clone BU20A, Thermo Fisher Scientific), anti-GFAP Alexa Fluor 647-conjugated antibody (51-9792-82; clone 2.2B10; Thermo Fisher Scientific), anti-nestin PE-conjugated antibody (MA5-23574; clone: 307501; Thermo Fisher Scientific) and anti-SOX2 Alexa Fluor 405-conjugated antibody (IC2018V, clone: 245610, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Techniques: BrdU Incorporation Assay, Cell Culture, Western Blot, Expressing, Concentration Assay
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