Heat inactivated calf serum

EL4 (ATCC® TIB-39™), derived from lymphoma in C57BL/6 N mouse, was purchased from ATCC. EL4 cells were cultured in DMEM (GIBCO Invitrogen) supplemented with 50 IU/mL penicillin, 50 mg/mL streptomycin (GIBCO Invitrogen) and 10% heat-inactivated calf serum (Sigma, USA). Cultures were maintained by replacement of fresh medium every 3 days, and cell density was kept between 1 X 10⁵ and 1 X 10⁶ cells/mL.

Bovine serum albumin (BSA), NADPH, human glutathione reductase (hGR), oxidized glutathione (GSSG) and 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), IMDM medium (Iscove’s Modified Dulbecco’s Medium), sodium bicarbonate, hypoxanthine, thymidine, bathocuprione sulfonic acid, cysteine, β-mercaptoethanol, heat-inactivated Calf serum, triton-X 100, anti-His tag antibody and protease inhibitors cocktail were purchased from Sigma-Aldrich (St. Louis, USA); oxidized trypanothione (TS₂) was purchased from Bachem (Bubendorf, Switzerland); NADPH-Glo kit and CellTiter-Glo were purchased from Promega (Madison, WI).

Giardia lamblia trophozoites (strain WB, ATCC 30957) were grown in modified TYI-S-33 medium (2% casein digest, 1% yeast extract, 1% glucose, 0.2% NaCl, 0.2% l-cysteine, 0.02% ascorbic acid, 0.2% K₂HPO₄, 0.06% KH₂PO₄, pH 7.1) supplemented with 10% heat-inactivated calf serum (Sigma-Aldrich) and 0.5 mg/mL bovine bile at 37°C (58 (link)). The cells were grown in 15-mL tubes with a screw cap with 5% CO₂ to 80% confluence (approximately 72 h) before 50 μL of the culture was transferred into 10 mL of fresh medium. Transfected Giardia strains were cultured in TYI-S-33 medium containing the appropriate antibiotics at the following concentrations: 50 μg/mL puromycin, 600 μg/mL G418, and 75 μg/mL blasticidin.
Giardia trophozoites (1 × 10⁵ cells/mL) were cultured in modified TYI-S-33 medium to 70 to 80% confluence. A portion of these cells were treated with 100 nM nocodazole (Sigma-Aldrich) for 2 h and harvested as G2/M-phase cells.