Trypanosoma brucei serum resistance and osmotic shock

Monomorphic Trypanosoma brucei brucei 2T1 bloodstream forms^{49 (link)} were cultured in HMI-11 [HMI-9 (GIBCO) + 10% v/v foetal bovine serum (GIBCO), Pen/Strep solution (penicillin 20 U ml⁻¹, streptomycin 20 mg ml⁻¹)] at 37 °C/5% CO₂ in vented flasks. Selective antibiotics were used as follows: 0.2 μg ml⁻¹ puromycin, 0.5 μg ml⁻¹ phleomycin (InvivoGen), 2.5 μg ml⁻¹ hygromycin B (Calbiochem). RNAi was induced in vitro with tetracycline (Sigma Aldrich) in 70% ethanol at 1 μg ml⁻¹.
To test for serum resistance 200 μl of log-phase parasites in HMI11 at 10⁶ cells ml⁻¹ were exposed to fresh rat serum for 3 h (10% and 50% concentration). Relative survival was measured of induced (for 48 h) and uninduced clones compared to no-serum controls. The whole experiment was performed in triplicate.
To test for response to osmotic shock^{18 (link)} 2 × 10⁶ cells were resuspended for 5 min at 4 °C in 0.5 ml of a solution 55 mM KCl/1 mM glucose to induce cell swelling. 0.5 ml of a 263 mM KCl/1.75 mM Mg₂Cl shrinking solution was added on top and cells incubated for 5 min at 4 °C. Remaining cells were washed once and resuspended in 1 ml of HMI11 media before counting. Survival of the induced cells compared to uninduced controls was plotted and significant results (p < 0.05) after T-tests indicated with a star.