Short-chain fatty acid effects on T-cell-mediated cancer cell killing

Activated T cells (5 × 10⁵ cells) were mixed with cancer cells (5 × 10⁴ cells) that were previously seeded (at least 2 h before) in 6-well plates in iMediam for T medium or RPMI-1640 supplemented as the above medium in a total volume of 2 mL. After another 2 h, SCFAs (acetic, propionic, butyric, isobutyric, valeric, isovaleric, or hexanoic acids) (Merck KGaA, Darmstadt, Germany) were added to the co-culture in increasing concentrations (0.1, 3, or 10 mM). In order to make a pH similar with that of 10 mM isobutyric acid treatment, hydrochloric acid was added at a final concentration of 10 mM. The co-culture was maintained at 37 °C in 5% CO₂ atmosphere for 72 h. The absolute numbers of cancer cells and T cells were determined using flow cytometry using fluorescent counting beads (Flow-Count, Beckman coulter, Brea, USA). After collecting floating cells, adherent cells were trypsinised and collected into the same tube. Fluorescent counting beads were added to each sample tube before staining cells with FITC or BV650-conjugated anti-CD45 antibody. CD45-positive cells were defined as T cells and all others as cancer cells. The numbers of T cells and cancer cells were corrected by the number of fluorophore beads counted in each sample (Supplementary Fig. S2).