T. cruzi culture

Epimastigote of T. cruzi Tulahuen strain (provided by Dr. Takeshi Nara, Juntendo University) was maintained in liver infusion tryptose (LIT) medium supplemented with 10% heat-inactivated FBS at 28°C. Metacyclogenesis was performed in RPMI medium [21], and differentiated metacyclic trypomastigotes were isolated by DEAE ion-exchange chromatography[22]. Metacyclic trypomastigotes were added to the 3T3-Swiss Albino fibroblast cell culture for infection, and amastigote-containing culture was maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin at 37°C under 5% CO₂ in a humidified incubator, until tissue-derived trypomastigotes emerged out to the culture supernatant.

Axenic amastigotes were obtained by in vitro amastigogenesis according to the method described by Tomlinson et al.[7] The tissue-derived trypomastigotes were collected from culture supernatant by centrifugation at 2000 ×g for 15 min, and were transformed into amastigotes by incubation in DMEM buffered with 20 mM MES (pH 5.0) and supplemented with 0.4% bovine serum albumin (BSA) for 24 h at 37°C.

To obtain intracellularly-derived amastigotes, infected 3T3 cells were detached from a culture flask by trypsin treatment, and centrifuged at 100 ×g for 5 min. The cells were washed by PBS to remove trypomastigotes and EA, and were suspended in 1 mL of Phosphate Saline Glucose buffer (1:9 mixture with 1% glucose)[23]. To release intracellular amastigotes, the cell suspension was passed through a syringe with 27 G needle 40 times to lyse the host 3T3 cells. Unbroken host cells and debris were removed by centrifugation at 100 ×g for 3 min. The intracellular amastigotes in the supernatant were purified by anion-exchange chromatography[23].