Sdm 79

Mid-log phase PCF T. brucei cultures were inoculated in SDM-79 (GIBCO) at density of 5 x 10⁵ cells.mL^-1 in the absence or presence of 2.5 μM or 5 μM N-methyl-N’-nitro-N-nitrosoguanidine (MNNG, Tokyo chemical industry Ltd). After 72 hours survival was determined using a haematocytometer. T. cruzi epimastigote form cells in the exponential growth phase were counted and diluted to 1x 10⁷ cells.mL^-1 in LIT medium in the absence or presence of 5 μM MNNG (Tokyo chemical industry Ltd). After 72 hours cell densities were determined by counting live cells with a haematocytometer using Erythrosin B exclusion.
Mid-log phase T. brucei PCF cells were diluted to 5 x 10⁵ cells.mL^-1 in SDM-79 (GIBCO) and then incubated with 10 μM or 20 μM H₂O₂ (VWR) at 27°C for 48–72 hours. BSF cells were diluted to 1 x 10⁶ cells.mL^-1 in HMI-9 medium (GIBCO) and incubated with 100 μM or 200 μM H₂O₂ (VWR) at 37°C, 5% CO₂ for 48 hours. After growth, cell density was measured using a haematocytometer. T. cruzi epimastigote form in exponential growth phase were diluted to 1x 10⁷ parasites.mL^-1 in PBS 1x and incubated with 75 μM H₂O₂ (Sigma-Aldrich) for 20 minutes at 28°C. After this, the cells were centrifuged, washed once with PBS and allowed to recover in LIT medium for 48 hours before cell densities were determined by counting live cells with a haematocytometer using Erythrosin B exclusion.

Bloodstream form (BSF) Trypanosoma brucei brucei strain MSUS/CI/78/TSW 196 [26] (link) was transformed into procyclic culture forms (PFs) by suspension in DTM medium with a final cis-aconitate (Sigma) concentration of 3 mM and cultured at 26°C [27] (link). Transformed trypanosomes were initially subcultured and maintained in a 1∶1 mix of DTM and SDM-79 media (Gibco), but subsequently cultured with SDM-79.

T. brucei cultures of the Lister 427 strain were maintained as both bloodstream (BSF) and procyclic (PCF) forms. BSF parasites were maintained at 37°C, 5% CO₂ in HMI-9 (GIBCO) medium supplemented with 20% fetal bovine serum (GIBCO). Cell passages were performed every 48 hours, with population density maintained between 1 x 10⁵ and 2 x 10⁶ cells.mL^-1. PCF cells were maintained at 27°C in SDM-79 (GIBCO) medium supplemented with 10% fetal bovine serum (GIBCO). Weekly passages were performed, but cell density was never lower than than 5 x 10⁵ cells.mL^-1.
Epimastigote forms of the CL Brener clone of T. cruzi were maintained in logarithmic growth phase at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum (GIBCO) and penicillin (10,000 U.mL^-1)/Streptomycin (10,000 μg.mL^-1) (GIBCO) as described by [41 (link)]. Metacyclic trypomastigotes, obtained after metacyclogenesis of epimastigotes cultures maintained in LIT medium for 15 to 20 days, were used to infect Vero cells cultured in DMEM medium (GIBCO) supplemented with 5% fetal bovine serum (GIBCO).