Cell culture of SH-SY5Y cells

SH-SY5Y cells (passage 50–70) were cultured as previously described (Attoff et al. 2016) and monthly screened for mycoplasma contamination (Lonza MycoAlert Mycoplasma Detection Kit). Briefly, they were cultured in MEM supplemented with 10% fetal bovine serum (Gibco, 31330095), 1% non-essential amino acid solution (Gibco, 11140035), 2 mM l-glutamine (Gibco, 25030024), 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, 15140122). For maintenance culture, SH-SY5Y cells were seeded at 27,000 cells/cm² in 75 cm² cell culture flasks (Corning). The cells were passaged once a week using TrypLE Express Enzyme (Gibco). SH-SY5Y cells were differentiated into a neuronal-like phenotype by exchanging the maintenance medium with differentiation medium consisting of DMEM/F12 (Gibco, 31330095) supplemented with 1 mM l-glutamine (Gibco, 25030024), 100 μg streptomycin/ml, 100 U penicillin/ml, 1 × N2-supplement (Gibco, 17502048) and 1 µM all-trans retinoic acid (RA, Sigma, R2625) 24 h after seeding. The cells were incubated in 100% humidity at 37 °C in air with 5% CO₂.

For determination of cell viability, 35,000 SH-SY5Y cells/well (109,375 cells/cm²) were seeded in maintenance culture medium in clear 96-well plates (Corning, 3599). 24 h after seeding, maintenance medium was replaced with differentiation medium and incubated for 3 days before exposure with compounds. Cell viability was determined after 24-h exposure with nicotine or neonicotinoids by the conversion of resazurin (Sigma, R2625) to resorufin in metabolically active cells (O’Brien et al. 2000). A 20 × resazurin stock solution was prepared by dissolving 11 mg resazurin salt in 1 ml 0.1 M NaOH and adjusting to 50 ml with PBS^−/− (pH set to 7.8 with 0.1 M HCl). The 20 × resazurin stock solution was sterile filtered and stored at 4 °C protected from light. After exposure with compounds, 150 μl medium was removed leaving 50 μl in each well and subsequently, 50 μl of 2 × resazurin solution dissolved in DMEM/F12 was added. The plate was incubated for 3 h at 37 °C with 5% CO₂. Resorufin fluorescence was measured at excitation 540 nm and at emission 590 nm using a FlexStation II fluorometer (Molecular Devices).