Pregs

Freshly isolated NK cells (4.5 × 10⁶ cells) were stimulated with 20 IU/ml of recombinant human IL-2 (specific activity 5 × 10⁶ IU/mg) (Miltenyi Biotech, BG, Germany). NK cells (7.5 × 10⁵ cells/condition) were also treated with the following drug combinations: (i) 30 µM PregS (Tocris Bioscience, Bristol, UK); (ii) 30 µM PregS and 3 µM Ononetin (Tocris Bioscience, Bristol, UK). These drug concentrations were determined using dose response analysis (Additional file 1: Figs. S3 and S4). NK cells were stimulated with IL-2 and treated with the above-mentioned drug combinations for 24 h at 37 °C with 5% CO₂ in Roswell Park Memorial Institute Medium (RPMI)-1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA). The treatment of NK cells with PregS and Ononetin for 24 h was to correspond with recommended duration of pre-activation of NK cells by cytokines.

The endogenous TRPM3 agonist PregS, the TRPA1 agonist cinnamaldehyde (CA), the TRPV1 agonist Caps, and the exogenous TRPM3 agonist CIM0216 were obtained from Tocris Bioscience (Bristol, UK). The TRPM3 antagonist Isosakuranetin (Isok) was obtained from Carl Roth (Karlsruhe, Germany). The well-characterized pruritogen Hist was obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA); and the non-histaminergic endogenous pruritic mediators 5-HT and ET-1 were purchased from Abcam (Abcam, Cambridge, MA, USA) or Sigma Aldrich.