EC50-Determination of RSL3 towards bloodstream T. brucei

1. Dilute logarithmically growing BS WT T. brucei in HMI-9 medium to a density of 2500  cells/ml. Fill each well of a 96-well plate with 90 µl of the diluted cell suspension.
2. Prepare a 10 mM stock solution of (1S, 3L)-RSL3 and RSL3 racemate in DMSO.
3. Using a separate 96-well plate, prepare dilutions of the compounds. You will need one row for the dilution series of one compound. Fill the first well of each row with 100 % DMSO, and the last well with 3 % DMSO in HMI-9. In a separate tube, pre-dilute the 10 mM stock solution of the compounds with HMI-9 medium to a 300 µM solution (which corresponds to a DMSO content of 3 %). Fill in the second well of the row at least 100 µl of this solution. Subsequently, use this solution for 9 consecutive steps of a serial 1:3 dilution, mixing 50 µl compound solution with 100 µl HMI-9 medium in wells 3 to 11.
4. Add 10 µl of each solution to the cells in the first 96-well plate (see step 1). This results in ten wells with cells and the compound in concentrations ranging from 30 µM to 1.4 nM, flanked by one well with 10 % DMSO and one well with 0.3 % DMSO, respectively (10 % DMSO serves as a lethal control and 0.3 % as a control of the highest final DMSO concentration in the cells treated with the compound). This step should be performed in triplicate. Two compounds can be tested on one plate leaving two rows on each plate which should contain only cells with no compound/DMSO.
5. Prepare one 96-well plate for each time point, that is 24, 48 and 72 h. Incubate the plates at 37 °C, in a 5 % CO₂ atmosphere.
6. After the respective cultivation times, add 50 µl ATPlite one step solution (PerkinElmer, Rodgau, Germany) to each well and immediately measure luminescence using a plate reader instrument (e. g. PerkinElmer VictorX4).
7. Plot the luminescence intensities values against the logarithmic compound concentration and calculate the IC50-values using e. g. GraphPad Prism software.
8. As control for the assay use chlorhexidine, a known trypanocidal compound and inhibitor of recombinant trypanothione reductase (Meiering et al., 2005; Beig et al., 2015). Prepare a 10 mM stock of the compound in DMSO. Perform 9 consecutive chlorhexidine dilutions similarly to as described in step 3, however starting with 370 µM and diluting first 1:9, followed by a 1:3 dilution. The subsequent dilutions should all be in 1:2 steps until the last concentration of 0.109 µM is reached. Follow the procedure described in points 4-7. The final chlorhexidine concentrations thus span the range from 37 µM to 11 nM.

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