Isopentenyl pyrophosphate ipp

PBMC from healthy donors were isolated from fresh buffy coats by Lympholyte (Cedarlane) density gradient centrifugation. Cells were cultured at a concentration of 4×10⁶/ml in the presence of IL-2 (6.5 U/ml), IL-15 (10 ηg/ml) (both from Sigma-Aldrich), TGF-β (1.7 g/ml; Calbiochem) and isopentenyl pyrophosphate (IPP; 20 µg/ml; Sigma). On days 3, 6, and 9, half of the supernatant volume was discarded and replaced with fresh medium containing cytokines. Aliquots of PBMC and bead-sorted γδ T cells (Miltenyi Biotec) were used fresh or were frozen for use at later time points.

PBMCs (2 × 10⁵ cells/well in triplicate) were stimulated with either uninfected RBCs (uRBCs) or iRBCs (6 × 10⁵/well) for 24 h or isopentenyl pyrophosphate (IPP, 3 μM, Sigma-Aldrich, St Louis, MO, USA) for 16 h. Brefeldin A (10 μg/ml, Sigma-Aldrich) and monensin (BD Biosciences) were added to the cells for the last 8 h of incubation. Assessment of γδ T cell cytokine production was performed by intracellular cytokine staining using allophycocyanin (APC)-conjugated anti-IFN-γ (clone B27, BD Biosciences) and PE-Cy7-conjugated anti-tumor necrosis factor alpha (TNF-α) (clone MAb11, eBioscience, San Diego, CA, USA), and cytotoxic capacity was assessed by Brilliant Violet 421-conjugated anti-CD107a (clone H4A3, Biolegend) staining in culture. A positive response was determined as the frequency of responding cells, which was twice above background and was ≥0.1% IFN-γ, TNF-α, or CD107a-positive γδ T cells of all γδ T cells or ≥0.5% positive γδ T cells of γδ T cell subsets following subtraction of background.

(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), capsaicin, savory oil, boric acid, fipronil sulfone and hydrochloric acid were purchased from Sigma-Aldrich (St. Louis, Missouri, USA); isopentenyl pyrophosphate (IPP), from Sigma-Aldrich (Tampa, USA, LC). The anti-coagulant/preservative, citrate-phosphate-dextrose-adenine was purchased from Vacuette (Greiner Bio-One Kremsmünster, Austria). 4-Aminobenzoic acid was purchased from Sigma-Aldrich Sweden (Stockholm, Sweden).