Fig. 2
Automated measurement of HuC/D expression and quantification of neurite outgrowth during neural differentiation. hNP cells were seeded onto 96 well plates at a density of 15,000 cells/well, differentiating hNP cultures were fixed at different time points for analysis following immunocytochemistry for HuC/D, βIII-tubulin antibody and nuclear staining. Cells were then imaged and quantified by Cellomics ArrayScan VTI HCS reader high-content imaging system. a–f and h–j represent cells at DIV 14. A, B (channel 1): Nuclei stained with Hoechst 33342, live cell nuclei (blue trace). c, d (channel 2): HuC/D+ cells stained (red trace), rejected cells stained (yellow trace). e, f: Pseudocolored composite image combining channels 1 and 2. b, d, f are magnified images to illustrate tracing in panel a–c. Scale bars = 50 μm. g: HuC/D+ expression throughout differentiation of neuronal cells. *Concentration is significantly different from control group (P < 0.05, one-way ANOVA), #Concentration is significantly different between groups (P < 0.05, one-way ANOVA). h (Channel 1): Nuclei identification. Blue trace = accepted, Yellow trace = rejected. I (Channel 2): Cell body masks based on βIII-tubulin and HuC/D expression; Blue trace = accepted cell, Red trace = rejected cell, Purple line = neurite, Yellow dot = branch point. Cells marked as rejected are not included calculating neurites per neuron or neurite length per neuron. Neurites emerging from accepted cell bodies are traced (purple lines) and quantified. j: Pseudo colored images from c and d merged. Scale bars = 50 μm