Par antibody

T. brucei procyclic strain 29–13 [29 (link)] was cultured at 28 °C in SDM-79 (Bioscience) containing 10 % (v/v) FCS and 0002 % hemin. T. brucei bloodstream strain 427 90-13 [29 (link)] was cultured at 37 °C in HMI-11 (Iscore’s Modified Dulbecco’s Medium (Invitrogen), 100 mg/L sodium pyruvate, 136.1 mg/L hypoxantine, 38.7 mg/L thymidine, 28.22 mg/L bathocuproinedisulfonic acid, 181.8 mg/L L-cysteine, 3.024 mg/L sodium carbonate, 196 μM β-mercaptoethanol) containing FCS (10 % v/v). Parasite viability was analyzed by microscopy.
For in culture inhibition assays, parasites of the procyclic form of Trypanosoma brucei were grown for 48 h up to a density of 5×10⁶ parasites/mL. Parasites of the bloodstream form were grown for 24 h up to a density of 5×10⁵ parasites/mL. In both cases, cells were harvested and preincubated for 30 min or 10 min in PBS-Glucose 2 % with inhibitors added, after which the parasites were treated with 500 μM or 250 μM hydrogen peroxide for procyclic and bloodstream forms, respectively, for 10 min. Protein extracts were prepared as indicated in our previous work [17 (link)], and 3 μg of total protein were manually spotted onto a membrane of nitrocellulose (GE Healthcare) for Dot blot analysis revealed with by commercial PAR antibody (BD).