Bactocasitone medium

To test the amoebicidal activity of the compounds, two strains of Naegleria fowleri (ATCC^® 30808™ and ATCC^® 30215™) of the American Type Culture Collection (LG Promochem, Barcelona, Spain) were used. The strain was axenically cultured at 37 °C in 2% (w/v) Bactocasitone medium (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% (v/v) foetal bovine serum (FBS), containing 0.5 mg/mL of streptomycin sulfate (Sigma-Aldrich, Madrid, Spain) and 0.3 µg/mL of Penicillin G Sodium Salt (Sigma-Aldrich, Madrid, Spain). Strains were kept in the biological security facilities level 3 of our institution following Spanish biosafety guidelines for this pathogen.
For the toxicity assays, the murine macrophage J774A.1 (ATCC # TIB-67) cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM, w/v), supplemented with 10% (v/v) fetal bovine serum and 10 µg/mL gentamicin (Sigma-Aldrich, Madrid, Spain), at 37 °C and 5% CO₂ atmosphere. For the experiments, all the strains were used during the logarithmic phase of growth.

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 (link)].

Two American Type Culture Collection strains of Naegleria fowleri, ATCC 30808 and ATCC 30215, were used to perform in vitro evaluation of the compounds (LG Promochem, Barcelona, Spain). Amoebae were axenically grown at 37 °C in 2% (v/w) Bacto Casitone medium (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% (v/v) fetal bovine serum (FBS) (Biowest, VWR, Barcelona, Spain), 0.3 μg/mL of Penicillin G Sodium Salt (Sigma-Aldrich, Madrid, Spain), and 0.5 mg/mL of streptomycin sulphate (Sigma-Aldrich, Madrid, Spain). Naegleria fowleri cultures were conserved in a biological security facility of level 3 at the Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias following the Spanish government’s biosafety guidelines for this pathogen with the minimum number of passages possible to avoid loss of pathogenicity.
Cytotoxicity assays were performed with a murine macrophage cell line (ATCC^® TIB-67) grown in Dulbecco’s Modified Eagle’s medium (GIBCO, Thermo Fisher Scientific) (DMEM, w/v) supplemented with 10% (v/v) FBS and 10 μg/mL of gentamicin (Sigma-Aldrich, Madrid, Spain). Cells were cultured in 5% CO₂.