Leishmania donovani 1S2D stress response assays

L. donovani promastigotes were cultivated in complete M199 media (pH 7.4) at 27 °C until they reach the stationary phase. For heat tolerance, promastigotes were incubated at 37 °C. To test their sensitivity to acidic pH, promastigotes were transferred to a pH 5.0 medium (same as the complete M199 medium except that the pH was adjusted to 5.0 using hydrochloric acid). For starvation response, promastigotes were transferred to PBS (pH 7.4). For resistance to oxidative or nitrosative stress, parasites were incubated in various concentrations of H₂O₂ or S-nitroso-N-acetylpenicillamine (SNAP). Cell viability was determined at the indicated times by flow cytometry after staining with 5 μg/ml of propidium iodide. Parasite growth was monitored using a Beckman Z2 Cell Counter.