Indole 3 pyruvic acid

Chlamydomonas wild-type and lao1 mutant were pre-cultured as described above to exponential phase. Cells were harvested by centrifugation for 2-3 min at 2,090 x g and washed three times with nitrogen-free TAP medium (T-N). Cells were subsequently incubated in T-N medium supplemented with 5 mM of L-Trp under continuous light and agitation (120 rpm). After 24 and 48 hours, cell-free supernatants were stored at -20°C until analyzed. L-Trp, indole-3-pyruvic acid and indole-3-acetic acid analysis was performed by the Chromatography Department staff at the Central Service for Research Support (SCAI) of the University of Córdoba, using High-Performance Liquid Chromatography (HPLC) and UV-Vis detection. A mixture of L-Trp (Sigma-Aldrich, Spain), indole-3-pyruvic acid (Sigma, Spain) and indole-3-acetic acid (GoldBio, US) was included as standard.

Prior to use in studies, macrophage-like RAW264.7 cells^{17 (link)} were cultured in 100 mm² plates (Sterilin Ltd., Newport, Gwent, UK) at 37 °C in a humidified 5% CO₂/air atmosphere in 18 mL complete RPMI media (RPMI-1640, 5% heat-inactivated foetal bovine serum, 200 mM GlutaMAX, 5000 μg/mL penicillin–streptomycin; ThermoFisher Scientific, Paisley, UK). Cells were passaged every 3–4 days when they had reached a density of approximately 2 × 10⁶ cells/10 mL medium.
For co-culture experiments, RAW264.7 cells (1 × 10⁵) were stimulated with 10 µg LPS (or PBS as a control) and co-cultured with 1 × 10⁶ STIB247 trypanosomes per well in 24 well tissue culture plates (ThermoFisher Scientific). In some experiments indolepyruvate (indole-3-pyruvic acid; Sigma-Aldrich) dissolved in absolute ethanol was included at a final concentration of 1 mM. Triplicate wells were used for each treatment group. The plates were incubated for 24 h at 37 °C in a humidified 5% CO₂/air atmosphere. The number of viable trypanosomes in each well was then counted using a haemocytometer. Experiments were repeated at least three times on separate days.

Indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA) were purchased from Sigma-Aldrich (98% minimal purity). Bacterial strains are listed in Supplementary Table S1, whereas plasmids and primers are listed in Supplementary Tables S2 and S3, respectively. Serratia strains were routinely grown at 25°C, unless otherwise indicated, in either Luria-Bertani (LB) broth or minimal medium (0.1% (w/v) (NH₄)₂SO₄, 0.41 mM MgSO₄, 40 mM K₂HPO₄, 14.7 mM KH₂PO₄, pH 7.0) supplemented with 15 mM glucose.