Permanox 8 well chamber slides

Neurite outgrowth assays were carried out on Permanox^© 8-well chamber slides (Thermo Scientific™). WT, α9-hNPCs and GFP-hNPCs were cultured on 20 μg/mL PLO and 10 μg/mL chicken TN-C (n = 4) or varying concentrations of human TN-C (Millipore): 1 μg/mL (n = 3), 5 μg/mL (n = 3) or 10 μg/mL (n = 3). Cells were plated at a density of 5 × 10⁴/well and incubated for 72 h at 37°C. The cells were fixed using 4% PFA and stained using the primary antibodies anti-GFP and anti-βIII-tubulin. Analysis and tracing of neurite outgrowth was performed using NeuronJ (Meijering et al., 2004 (link); Schneider et al., 2012 (link)) and statistical analysis was performed using Microsoft Excel and Prism 5 GraphPad. A total of 30 neurite/axon lengths were measured for each condition. One replicate consisted of one vial of hNPCs cultured in one chamber slide (with 2 wells/condition). Data sets were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc.

HeLa cells were seeded at 5000 cells/cm² in serum growth media onto a permanox 8-well chamber slides (Thermo Fisher, VIC, Australia), cultured under normal conditions in an incubator overnight. After 24 h incubation, medium was replaced with reduced serum media (opti-MEM) and equilibrated for 20 min. Cells were then treated with either one of the polymers or siRNA complexes (equivalent to nmol siRNA) conjugated with FITC fluorophores for 1 h at 37°C (as previously undertaken (26 (link), 28 (link))), after which the cells were washed three times with PBS to remove unbounded polymers/complexes. Cells were fixed with 4% para-formaldehyde for 20 min at room temperature, and later washed with PBS. Plasma red cell mask stain was then added to each well and incubated for an additional 20 min. Mountant was added prior to being covered with a glass coverslip, which was sealed and imaged immediately using an Olympus BX53 fluorescence microscope with Cellsens dimensions image software (Olympus Imaging Australia, NSW, Australia).