2.3. Amoebic Strains and Cell Line Maintenance

The American Type Culture Collection strains ATCC^® 30808™ and ATCC^® 30215™ of Naegleria fowleri (LG Promochem, Barcelona, Spain) were used for the activity assays. Amoebae were grown at 37 °C in axenic conditions in 2% (w/v) bactocasitone medium (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% (v/v) foetal bovine serum (FBS), 0.3 µg/mL of penicillin G and 0,5 mg/mL of streptomycin sulphate (Sigma-Aldrich, Madrid, Spain). As required by the Spanish Government biosafety guidelines for this pathogen, amoebic strains were cultured in a biological security facility level 3 at the Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna.

Evaluation of in vitro toxicity was carried out using the murine macrophage J774A.1 cell line (ATCC # TIB-67, LG Promochem, Barcelona, Spain) which was routinely cultured in Dulbecco’s Modified Eagle’s medium (DMEM, w/v) supplemented with 10% (v/v) FBS and 10 µg/mL of gentamicin (Sigma–Aldrich, Madrid, Spain), at 37 °C in a 5% CO₂ atmosphere as previously described [35].