Molecular probes neurite outgrowth staining kit

For MTT assays, 1 uM MG 132 (Tocris Bioscience, United Kingdom) was added to select wells containing 10,000 cells per well at 72 hr post-differentiation. Cell viability was assessed at 96 hr post-differentiation. Following manufacturer's protocol, CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega; Madison, WI) was used to determine cell number. Cells were incubated for 1.5 hr at 37°C and 5% CO₂ with MTT dye solution. Undifferentiated HD cells were serially diluted across a 96-well plate to create a standard curve for cell number calculation. Absorbance was measured using Bio-Tek Synergy H1 spectrophotometer at 540 nm for miR-10b-5p transfected wells, with MG 132 (n = 44) and without MG 132 (n = 35) and cel-miR-67-3p transfected wells with MG 132 (n = 40) and without MG 132 (n = 40). One-way ANOVA way used for statistical analysis.
For cell viability staining, miR-10b-5p and negative control mimic were transfected after 48 hours of differentiation in 12-well culture plate with 4 replicates each, 250,000 cells per well. Molecular Probes Neurite Outgrowth Staining Kit (Life Technologies, Carlsbad, CA) was used according to manufacturer's protocol. Using Bio-Tek Synergy H1 microplate reader, fluorescent area scans were taken at 530 nm excitation/590 nm emission with a 5×5 matrix per well.