Computational protocol: Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation

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Protocol publication

[…] After photoporation, the sample was placed back in the incubator for at least 1 h to allow the cells to recover and permit equal phalloidin distribution throughout the targeted neuron. After gently washing excess phalloidin in the culture medium, labeled cells were imaged with a confocal laser scanning microscope using both low (10 × NA 0.45) and high (60 × W NA 1.20) magnification objective lenses (λex = 488 nm, λem = 525 ± 25 nm or λex = 647 nm, λem = 700 ± 40 nm). To visualize the entire neuronal network, the membrane-permeable dye SiR-tubulin (1 μM final concentration) was added for 30 min prior to imaging (λex = 647 nm, λem = 705 ± 45 nm). Confocal images were usually acquired within 1 h after recovery from the photoporation procedure, but some cultures were imaged 24 h after SNAP. Quantification of dendritic spine density, i.e., the number of spines divided by the dendrite length, was done manually assisted by ImageJ software (Schneider et al., ). [...] For determination of cell viability with PrestoBlue, one isolation was considered with six wells per condition. For quantification of dendritic spine density, three independent isolations were considered. For each isolation, >1000 μm of dendrite stretches were analyzed on 10 selected neurons for each DIV or DMOG concentration.Statistical analyses were carried out in SAS JMP Pro 12 software, and the detailed results of those tests are reported in Table . Shapiro-Wilk tests were used to check for normality. Since some of the data sets were not normally distributed, non-parametric tests were performed throughout the article. To assess the overall effect, Mann-Whitney (2 groups) or Kruskal-Wallis rank sums (>2 groups) tests were performed. When assessing the influence of culture age or DMOG treatment on spine density, these tests were performed within the method group (SNAP or DiI). For PrestoBlue measurements, these tests were performed within the time group (2 or 24 h). Conditional to the overall Kruskal-Wallis test, post hoc tests were performed. To detect differences across DIVs, Dunn all pairs tests for joint ranks were used as the non-parametric alternative for Bonferroni tests (Dunn, ). For the DMOG experiment, a Steel test for comparison with control was used as the non-parametric alternative for a Dunnett test (Steel, ). The data are represented as bar charts (mean + standard deviation). […]

Pipeline specifications

Software tools ImageJ, JMP Pro
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Diseases Neurodegenerative Diseases