10 ml culture tubes

Trophozoites from G. lamblia WBC6 were grown under anaerobic conditions in 10 mL culture tubes (Nunc, Roskilde, Denmark) on a modified TYI-S-33 medium, as previously described [42 (link)]. Subcultures were performed by inoculating 100 μL of cells from a confluent culture detached by cooling to a new tube containing 10 mL of the culture medium as described [43 (link)]. Pellets were washed three times with ice-cold PBS, then counted and stored at −80 °C for subsequent size exclusion chromatography.

Trophozoites from G. lamblia WB clone C6 wild-type and of the NTZ/MET resistant clone C4 were grown under anaerobic conditions in 10 ml culture tubes (Nunc, Roskilde, Denmark) containing modified TYI-S-33 medium as previously described (Clark and Diamond, 2002 (link)). C4 was routinely cultured in the presence of 50 μM NTZ. Subcultures were performed by inoculating 20 μl (wild-type) or 100 μl (C4) of cells from a confluent culture detached by cooling (Müller et al., 2006 (link)) to a new tube containing the appropriate medium.

Trophozoites from G. lamblia WBC6 (C6), WBA1 (A1) and GS/M-83-H7 (H7) were grown under anaerobic conditions in 10 mL culture tubes (Nunc, Roskilde, Denmark) on modified TYI-S-33 medium as previously described (Clark and Diamond, 2002). In order to ensure the growth of the A1 and H7 clones, the components were as close as possible to the isolation medium, in particular heat-inactivated adult bovine serum (Biofluids, Rockville, MD, USA) and casein peptone (Becton Dickinson, Cockeysville, MD, USA). Prior to shotgun mass spectrometry analysis, the cultures were routinely passaged two times. Subcultures were performed by inoculating 100 μL of cells from a confluent culture detached by cooling (see below) to a new tube containing 10 mL culture medium (Müller et al., 2006). Trophozoites were harvested by incubation on ice for 15 min followed by centrifugation (300 × g, 10 min, 4°C). Pellets were washed three times with ice-cold PBS, counted and stored at −80°C for subsequent proteomic analysis or for enzymatic assays.