Computational protocol: Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

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Protocol publication

[…] hMSCs (passages 3–6) were fixed with 4% paraformaldehyde for 10 min, and were permeabilized with 0.02% Triton X-100 for 10 min, followed by blocking with 5% FBS for 1 h and incubation with primary antibodies for at least 1 h. The primary antibodies were as follows: rabbit anti-MAP2 polyclonal antibodies (1∶500; cat#AB5622, Chemicon); rabbit anti-synapsin-1 (SYN1) polyclonal antibodies (1∶500; cat#AB1543, Chemicon); mouse anti-choline acetyltransferase (ChAT) monoclonal antibodies (1∶500; clone 1E6, cat#MAB305, Chemicon); and rabbit anti-dopamine β-hydroxylase (DBH) polyclonal antibodies (1∶500; cat#AB1585, Chemicon); and mouse anti-β-catenin monoclonal antibodies (1∶500; clone 5H10, cat#MAB2081, Chemicon,). Cells were then incubated for 1 h with secondary antibodies, goat anti-mouse IgG:Dylight 488 (1∶100; Serotec), or sheep anti-rabbit IgG:Dylight 649 (1∶100; Serotec). The nuclear stain was 4',6-diamidino-2-phenylindole (DAPI) (Chemicon). Images were obtained using fluorescent microscopy (Hamamatsu EM-CCD Camera C9100-13). For β-catenin staining, cells with projections exceeding 30 µm were considered neurite-positive cells. The cell area and neurite length were calculated using ImageJ software. At least 1000 cells from 10∼15 viewing fields per group were used to calculate percentages of cells. The human SH-SY5Y neuroblastoma cell line was used as the positive control, and the human MG63 osteosarcoma cell line was used as the negative control. Mouse IgG1 antibodies (Chemicon) were used as the isotype control. All control results are shown in . [...] Data are presented as mean values ± SD of all experiments or a representative result of three or more experiments. Quantitative data were analyzed using SigmaPlot 9.0 or SPSS software by conducting either the Student's t test or a one-way ANOVA. A value of p<0.05 (*, #, 1, 2, 3, 4, 5 or 6) and p<0.01 (**, ##, 1″, 2″, 3″, 4″, 5″ or 6″) indicated significance. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Lithium