Tween 80

All reagents used in the lab procedures were of HPLC grade: hexane and sulphuric acid were obtained from Labscan (Dublin, Ireland), linoleic acid (C18:2 cis 9 cis 12) from Sigma-Aldrich (St. Louis, MO, USA), linolenic acid (C18:3 cis 9 cis 12 cis 15) from Nu-Chek Prep, Inc. (Elysian, USA), and high CLA content oil (Tonalin) from Cognis (Illertissen, Germany). LA and LNA were prepared as a 30000 μg mL⁻¹ stock solution containing 2% (w/v) Tween 80 (Scharlau, Sentmenat, Barcelona, Spain) and filter-sterilized through a 0.45 μm-pore size membrane (Sarstedt, Nümbrecht, Germany).

Two-fold serial dilutions of the compounds were prepared in 7H9 broth supplemented with 0.2% glycerol and 10% ADC in 96-well flat-bottomed plates and seeded with M. smegmatis at a bacterial density of 10⁵ CFU/mL. Plates were incubated for 3 h before addition of 30 µL of the bacterial growth reporter MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (2.5 mg/mL) (Sigma, Madrid, Spain) and Tween 80 (10%, v/v) (Scharlau, Barcelona, Spain). The optical density at 580 nm was measured after further 3 h incubation, the lowest compound concentration that inhibited 90% MTT conversion to formazan was used to define the MIC value.
M. tuberculosis strains were tested in duplicate in 96-well flat-bottomed plates in 7H9 with 10% OADC in a final volume of 200 µL. Bacteria were incubated for 7 days in the presence of the compounds before the addition of 10 µL of 0.025 mg/mL resazurin. Following 1-day incubation, compound activity was determined by fluorescence measures (530 nm excitation, 590 nm emission).