Nunc cell culture flask

Human Caucasian lung carcinoma cells (adenocarcinomic human alveolar basal epithelial, A549) were obtained from European Collection of Authenticated Cell Cultures (Public Health England, UK) and grown in 175 cm² Nunc cell culture flasks (ThermoFisher, Rugby, UK). Ham’s F-12K (Kaighn’s) Medium (F-12K) (Gibco, Paisley, UK) was supplemented with 10% USA-origin fetal bovine serum (FBS) purchased from Sigma-Aldrich (Dorset, UK), 100 units/mL penicillin, 100 μg·mL⁻¹ streptomycin, and 250 ng·mL⁻¹ amphotericin B (PSA) (HyClone, Cramlington, UK). A549 cells were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. General maintenance of the cell line was completed by passaging every 7 days or before reaching 90% confluency and renewing culture medium every 3–4 days. Cells were dissociated using a balanced salt solution containing trypsin (0.25%) and EDTA (1 mM) (Gibsco) and reseeded at a density of 1.87 × 10⁵ cells per 175 cm² cell culture flasks.

Vero cells (ECACC 84113001) were cultured in Nunc cell-culture flasks of 75 cm² surface area (Thermo Fischer Scientific, Waltham, MA, USA) in Minimum Essential Medium (MEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) previously inactivated at 56 °C for 30 min (iFBS), plus antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL). The cultures were maintained at 37 °C, in a moist atmosphere enriched with 5% CO₂.
Trypanosoma cruzi Pan4 strain (TcIa + TcId) was employed in this work. This strain was isolated from a patient from Panama during 2004. The epimastigote stage of the parasite was cultured in liver infusion tryptose (LIT) medium supplemented with 10% heat-inactivated fetal bovine serum, at 28 °C. Trypomastigote forms derived from cell cultures were obtained from infected Vero cell cultures as previously described [51 (link),52 (link),53 (link),54 (link)].

The human neuroblastoma SH-SY5Y cells were cultured in DMEM medium with high glucose supplement, 10% FBS, and 0.5% streptomycin-penicillin. They were incubated at 37 °C in a saturated humidity atmosphere containing 5% CO₂. Before every experiment, the cells were grown in plastic Nunc cell culture flasks (75 cm²) (Thermo Scientific, Illkirch, France) and were treated with 10 μM retinoic acid (RA) for 5 days towards differentiation into neuronal cells [19 (link),40 (link),71 (link),72 (link)]. The adherent SH-SY5Y cells were divided twice weekly with the use of 0.05% trypsin-EDTA for up to 5 min, followed by centrifugation (200× g) at 4 °C for 5 min. The cells were counted using a KOVA® cell counting chamber (VWR, Fontenay-sous-Bois, France), and seeded at densities of 2 × 10⁴ cells/well in 96-well plates or 10⁶ cells/flask (25 cm²) (depending on the type of biological analysis to be carried out). After 24 h, the SH-SY5Y cells were incubated with RA (10 μM) for 5 days, changing the medium with RA at least once. The neuronal phenotype was distinguished by the extensive proliferation of neurites [71 (link),72 (link)].