Figure 5

The labeling and tracking of neuronal differentiation of ECS cells using FNDs.

(a) A model of ECS and neuronal differentiation labeled with FND. (b) P19 cells were treated with or without FNDs (50 μg/ml for 24 h). Then the cells were differentiated into neuron cells by RA induction. The fluorescence intensity of FND in P19 and the differentiated neuron cells were measured by flow cytometer. The fluorescence intensity of FND was excited with wavelength 488 nm and the emission was collected by >650 nm signal range (FL3-H). (c) P19 cells were treated with 50 μg/ml FNDs for 24 h. Then the cells were differentiated into neuron cells by RA induction. The cell morphology of P19 cells and differentiated neuron cells were observed under a phase contrast microscope. (d) The P19 cells and differentiated neuron cells were incubated with mouse anti-SSEA-1 and rabbit anti-β-III-tubulin antibodies and then incubated with goat anti-mouse IgG Cy3 and goat anti-rabbit IgG HiLyte Fluor 488 antibodies. The nuclei were incubated with Hoechst 33258. The red fluorescence intensity of FND was excited with wavelength 580 nm and the emission was collected in 600–700 nm. The fluorescence intensity of nuclei was excited with 405 nm and the emission was collected in 415–485 nm. The fluorescence intensity of β-III-tubulin was excited with 488 nm and the emission was collected in 505–545 nm. The fluorescence intensity of SSEA-1 was excited with 543 nm and the emission was collected in 560–580 nm. The nuclei display blue color. The SSEA-1 is indicated as yellow color. The β-III-tubulin shows green color.