Heat inactivated adult bovine serum

E. histolytica strain HM-1:IMSS trophozoites were maintained in 50 ml culture flasks (Greiner Bio-One) containing TYI-S-33 media, 10% (v/v) heat-inactivated adult bovine serum (Sigma), 1% (v/v) MEM Vitamin Solution (Gibco), supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Omega Scientific) [20 (link)]. E. invadens strain IP-1 were cultured in LYI-S-2 at 25°C [32 (link), 33 ].

G. intestinalis cells (strains WB, ATCC 30957 and HP-1- Prague line of Portland-1 isolate ATCC 3088) were cultured in TYI-S-33 medium supplemented with 10% heat-inactivated adult bovine serum (Sigma-Aldrich), 0.1% bovine bile, and appropriate antibiotics at 37 °C [95 ]. The enrichment of mitotic cells was done according to [36 (link)]. Trophozoites from late log phase were incubated in growth medium supplemented with 100 ng/ml of albendazole (Sigma-Aldrich) for 6 h at 37 °C [36 (link)]. After the incubation, the albendazole-affected detached cells were discarded, and the unaffected adherent pre-mitotic cells were washed twice with pre-warmed fresh drug-free medium and then detached from the tube by cooling on ice for 10 min. The cells were then allowed to proliferate on slides in the drug-free conditions for 9–20 min and fixed by ice-cold methanol (see below) for fluorescence microscopy or by 2.5% glutaraldehyde (see below) for electron microscopy. For the analysis of cells with blocked mitosis, the albendazole-affected (detached) cells were fixed by 1% formaldehyde (see below).