Metaxpress neurite outgrowth application module

As described, ∆K280 tau_RD-DsRed SH-SY5Y cells were seeded in a 24-well plate (5 × 10⁴/well) with retinoic acid addition on day 1, treated with tested compounds (10 µM) and ∆K280 tau_RD-DsRed expression was induced with doxycycline (2 μg/mL) on day 2. On day 8, after being fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 for 10 min and blocked in 3% bovine serum albumin (BSA) for 20 min, the cells were stained with TUBB3 (neuronal class III β-tubulin) primary antibody (1:1000; BioLegend, San Diego, CA, USA) at 4 °C overnight, followed by goat anti-rabbit Alexa Fluor^® 555 secondary antibody (1:1000; Molecular probes) at room temperature for 2 h, with 4′-6-diamidino-2-phenylindole (DAPI, 0.1 µg/mL; Sigma-Aldrich) included for nuclei staining. Neuronal images were captured using the high-content analysis system as described. Neurite total length (μm), processes (primary neurite extensions projecting directly from the cell body) and branching (points at which primary neurites bifurcated) were analyzed using Neurite Outgrowth Application Module (MetaXpress; Molecular Devices). Around 5000 cells were analyzed in each of three independent experiments for each sample.

On day 1, Aβ-GFP or ∆K280 tau_RD-DsRed SH-SY5Y cells were plated into 24-well plates (6 × 10⁴/well) followed by addition of retinoic acid. On day 2, cells were treated with coumarin-chalcone derivatives (5 or 10 μM) and with doxycycline (5 or 2 μg/ml) to induce the expression of Aβ-GFP or ∆K280 tau_RD-DsRed. On day 8, 4% paraformaldehyde, 0.1% Triton X-100, and 3% BSA were used to fix, permeabilize, and block cells, respectively. Subsequently, the cells were stained with TUBB3 (neuronal class III β-tubulin) primary antibody (1 : 1000; Covance, Princeton, NJ, USA) at 4°C overnight. Goat anti-rabbit Alexa Fluor 555 secondary antibody (1 : 1000; Thermo Fisher Scientific) was then added, followed by a 3 h incubation at room temperature. Following staining nuclei with 4′,-6-diamidino-2-phenylindole (DAPI) (0.1 μg/ml; Sigma-Aldrich), the neurons were imaged by using the HCA system and analysed by using the Neurite Outgrowth Application Module (MetaXpress; Molecular Devices).