Ting typical baseline (R0) of your ratiometric measurements as described above for nonratiometric measurements. While expression levels of GCaMP2 varied from cell to cell, this did not affect 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 more energy spectral density analysis (Uhlen, 2004; Bortone and Polleux, 2009), which measures periodicity in a time series signal with out an arbitrary definition of a transient. This evaluation (our unpublished observations) confirmed higher periodicity as measured by average relative energy in calcium signals in contralateral vs. ipsilateral axons at a frequency of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 were performed as previously described (Li et al., 2009). Briefly, cortical neurons were dissociated from P0 hamster sensorimotor cortex and electroporated with EGFP-CaMKIIN plasmids with an Amaxa Nucleofector. These neurons have been plated onto coverslips coated with 0.5 mg mL poly-D-lysine (Sigma) and 20 lg mL laminin (Sigma/Invitrogen) at a density of CM10 medchemexpress 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 had been then incubated for 72 h before fixation. Axon lengths were measured in neurons expressing EGFP-CaMKIIN or in untransfected neurons from the identical dish as a manage.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 (ten k cells/well in a six well plate (Falcon). Assembly of your Dunn chamber (Hawksley, UK) was modified from previDevelopmental NeurobiologyHutchins et al. noted, comparisons among two groups had been created with Student’s t test and comparisons among a number of groups have been made having a one-way ANOVA with Dunnett’s posttest. Measurements are provided in mean 6 SEM unless 72178-02-0 Technical Information otherwise noted. Photos were modified with a low-pass filter in MetaMorph to lessen single-pixel noise. The images presented in figures had 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 technique [Fig. 3(C)].ous research (Yam et al., 2009). Dunn chambers had been rinsed by serum-free medium after then both inner and outer wells have been filled by serum-free medium. To secure coverslips with neurons on the chamber, silicon sealant (Dow Corning) was applied at 0.five cm from the border of outer nicely but omitted at one particular side to form a slit later for draining and refilling the outer properly. A coverslip with neurons was inverted over the Dunn chamber leaving a narrow slit in the edge without having the sealant. Media at the outer well was aspirated after which medium with 400 ng mL Wnt5a was added to the outer well. The narrow slit was sealed by fixing a tiny piece of parafilm (American National Can) to the chamber with sealant. Photos have been acquired straight away just after Dunn chamber assembly and 2 h later having a 20 three 0.five numerical aperture (NA).