Nunc easyflask 75 cm2 flasks

Acanthamoeba castellanii Neff (ATCC 30,010) cells were grown at RT in PYG (proteose peptone, yeast extract, glucose) medium in Nunc™ EasYFlask™ 75 cm² flasks (Thermo Scientific) and subcultured twice a week (dilution 1:5). Giardia lamblia WB C6 (ATCC 50,803) cells were grown in Keister`s modified TYI-S-33 medium containing bile [34 (link)] at 37 °C in completely filled Nunclon Delta tubes (Thermo Scientific) and subcultured every second day.

TF-1-CN5a.1 (product CRL-2512™, American Type Culture Collection, ATCC^®, Manassas, VA, USA) cells were obtained from LGC Standards (Teddington, UK). The cells were maintained in complete growth medium of Roswell Park Memorial Institute (RPMI) 1640 medium, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate (RPMI-1640, ATCC modification; Gibco^TM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco^TM, Thermo Fisher Scientific), 2 ng/mL human granulocyte macrophage colony-stimulating factor (hGM-CSF) (Sigma-Aldrich, St. Louis, MO, USA), 0.4 mg/mL G-418 (Calbiochem^TM, Merck), and 100 U/mL penicillin – 100 µg/mL streptomycin (Gibco^TM, Thermo Fisher Scientific). The cells were maintained as stationary suspension cultures in non-treated Nunc™ EasyFlask™ 75 cm² flasks (Thermo Fisher Scientific) in a fully humidified 5% CO₂ atmosphere at 37 °C. Cell number and viability were determined using trypan blue.