High-Content Screening of Neurotoxic Compounds

After 2 days of differentiation, 30000 LUHMES cells were reseeded into each well of a 96-well plate in DMed containing only tetracycline. After cells’ attachment for 1 h, they were exposed to FRs/solvent(s) for 24 h. One hour before read-out, cells were stained with Calcein-AM and H-33342 and imaged via a high-content imaging microscope (Cellomics ArrayScanVTI, Thermo Fisher Scientific) to assess neurite area. For neurite area determination, an automated algorithm was used, which calculates the area of the cell soma and subtracts this area from all calcein-positive pixels imaged (Stiegler et al. 2011 (link); Krug et al. 2013a (link)). To assess viability, all stained nuclei (H-33342 positive) are used to determine total cell number and H-33342 and calcein double-positive cells are defined as viable cells (Stiegler et al. 2011 (link); Krug et al. 2013a (link)). Each compound was tested in serial dilutions (1:3) with 10 concentrations starting at 20 μM and SC plated in three replica wells per condition. Effects of TBBPA, BDE-47, BDE-99, IDDPHP, TCP, t-BPDPHP, EHDPHP, TPHP, and IPPHP were assessed in a previous screening (Delp et al. 2018 (link)). TDCIPP, TOCP, and TCIPP were negative in a pre-screening at 20 μM and therefore not tested any further (data not shown).