alvetex protocol  (ReproCELL)

Journal: Molecular Neurobiology

Article Title: Exposure of the SH-SY5Y Human Neuroblastoma Cells to 50-Hz Magnetic Field: Comparison Between Two-Dimensional (2D) and Three-Dimensional (3D) In Vitro Cultures

doi: 10.1007/s12035-020-02192-x

Figure Lengend Snippet: Exposure of 3D-grown SH-SY5Y neuroblastoma cells to ELF-MF stimulates the differentiation into a dopaminergic (DA) phenotype. a Graphical sketch of the experimental schedule. SH-SY5Y cells were seeded in either 2D multi-wells or 3D Alvetex ® scaffold and treated as follows (DA differentiation + MF exposure): 3 days of RA administration (without any MF/sham exposure), followed by 3 days of PMA treatment (the latter 2 days in the presence of either ELF-MF or sham exposure). b The expression level of TH and DAT was assessed in both 2D and 3D growing conditions at the end of the differentiation treatment (RA + PMA, without any MF exposure) compared to the basal proliferating phenotype. Values are means ± SD ( N = 3 independent experiments); * P < 0.05, calculated in the RA/PMA-differentiated cells versus the proliferating ones (chosen as reference). Evaluation of the c relative proliferation index (by WST-1 assay), d percentage of cells populating the G 0 /G 1 phase of the cell cycle with e corresponding FACS histograms chosen as representative of three independent experiments. f Analysis of the p21 transcript expression level, carried out by real-time PCR (18s normalization). All analyses in panels c–f were performed at the end of the differentiation/exposure schedule. Values represent means ± SD ( N = 3 independent experiments); * P < 0.05. g Gene expression assessment of DAT , TH , GAP43 , TUBB3 , Nurr1 , and NeuN carried out by real-time PCR (18s normalization) in the RA/PMA-differentiated cells following exposure to either 50 Hz or sham field. Values are means ± SD ( N = 3 independent experiments); * P < 0.05

Article Snippet: Both H&E and immunohistochemistry (IHC)/immunofluorescence (IF) immunostaining have been carried out in paraformaldehyde-fixed 2D cultures and in formalin-embedded 3D slices, the latter according to Alvetex ® protocols ( https://www.reprocell.com ).

Techniques: Expressing, WST-1 Assay, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Molecular Neurobiology

Article Title: Exposure of the SH-SY5Y Human Neuroblastoma Cells to 50-Hz Magnetic Field: Comparison Between Two-Dimensional (2D) and Three-Dimensional (3D) In Vitro Cultures

doi: 10.1007/s12035-020-02192-x

Figure Lengend Snippet: 50-Hz MF does not affect proliferation, apoptosis, and angiogenetic biomarkers in proliferating SH-SY5Y cells grown in both 2D and 3D cultures. Cells were exposed for 72 h to 50-Hz (1 mT) MF and characterized according to the following endpoints: a relative proliferation index (by WST-1 test) and b cell cycle distribution (FACS analysis of DNA content by PI staining). Values represent means ± SD ( N = 3 independent experiments). c Evaluation of proliferative ability carried out by IF in 2D conventional cultures (merge Ki67/DAPI staining) and by IHC in 3D Alvetex ® scaffolds (PCNA staining). Scale bar: 200 μm. d Assessment of apoptosis percentage by active-caspase-3 antibody staining. Scale bar: 200 μm. e , f Gene expression analysis of proliferation, invasiveness, and neo-angiogenesis-related transcripts (normalized to 18s expression by real-time PCR), performed in 2D- versus 3D-cultured SH-SY5Y cells. The values reported represent the mean fold change (± SD) in mRNA expression of ELF- versus sham-exposed cells (chosen as a reference value). No significant difference has been observed ( N = 3 independent experiments)

Article Snippet: Both H&E and immunohistochemistry (IHC)/immunofluorescence (IF) immunostaining have been carried out in paraformaldehyde-fixed 2D cultures and in formalin-embedded 3D slices, the latter according to Alvetex ® protocols ( https://www.reprocell.com ).

Techniques: Staining, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Cell Culture