Ting typical baseline (R0) in the ratiometric measurements as described above for nonratiometric measurements. Despite the fact that expression levels of GCaMP2 varied from cell to cell, this did not influence the frequency of calcium transients reported. Raw baseline fluorescence did not correlate with frequency (Spearman correlation coefficient r 0.09, p 0.69). We validated our calcium transient measurements with added power spectral density analysis (Uhlen, 2004; Bortone and Polleux, 2009), which measures periodicity in a time series signal with no an arbitrary definition of a transient. This analysis (our unpublished observations) confirmed larger periodicity as measured by average relative energy in calcium signals in contralateral vs. ipsilateral axons at a frequency of 15 per hour, the frequency of calcium transients evoked by Wnt5a in vitro (Li et al., 2009).Dissociated Cortical Neuron Cultures and Wnt5a ExperimentsCulture of dissociated cortical neurons and bath application experiments with Wnt5a have been performed as previously described (Li et al., 2009). Briefly, cortical neurons have been dissociated from P0 hamster sensorimotor cortex and electroporated with EGFP-CaMKIIN plasmids with an Amaxa Nucleofector. These neurons have been plated onto coverslips 1456632-40-8 Autophagy coated with 0.5 mg mL poly-D-lysine (Sigma) and 20 lg mL laminin (Sigma/Invitrogen) at a density of 20007000 per cm2 and had been incubated in five CO2 and 9 O2 at 378C for two days. For long-term axon outgrowth assays, 400 ng mL Wnt5a in 0.5 BSA is PBS, or BSA alone, was then added to the cultures. Cultures have been then incubated for 72 h ahead of fixation. Axon lengths had been measured in neurons expressing EGFP-CaMKIIN or in untransfected neurons in the similar dish as a control.Dunn Chamber Axon Guidance Assay and AnalysisFor Dunn chamber axon guidance assays, P0 hamster cortical neurons have been grown on appropriately coated (see above) 22-mm2 No. 1.five coverslips (Corning) at a low density (10 k cells/well inside a six nicely plate (Falcon). Assembly on the Dunn chamber (Hawksley, UK) was modified from previDevelopmental NeurobiologyHutchins et al. noted, comparisons in between two groups had been produced with Student’s t test and comparisons involving numerous groups were produced having a one-way ANOVA with Dunnett’s posttest. Measurements are offered in mean six SEM unless otherwise noted. Pictures have been modified using a low-pass filter in MetaMorph to lessen single-pixel noise. The images presented in figures have been enhanced with brightness-contrast adjustments in Adobe (Mountain View, CA) Photoshop, and with Flatten Background and Sharpen adjustments in MetaMorph for slice pictures taken from the Nikon epifluorescence program [Fig. three(C)].ous studies (Yam et al., 2009). Dunn chambers had been rinsed by serum-free medium once and then each inner and outer wells have been filled by serum-free medium. To secure coverslips with neurons around the chamber, silicon sealant (Dow Corning) was applied at 0.5 cm in the border of outer well but omitted at a single side to kind a slit later for draining and 2628-17-3 Technical Information refilling the outer effectively. A coverslip with neurons was inverted over the Dunn chamber leaving a narrow slit at the edge devoid of the sealant. Media in the outer well was aspirated then medium with 400 ng mL Wnt5a was added for the outer well. The narrow slit was sealed by fixing a small piece of parafilm (American National Can) to the chamber with sealant. Pictures had been acquired straight away after Dunn chamber assembly and 2 h later having a 20 3 0.five numerical aperture (NA).
