Rpmi medium

Experiments to analyze changes in calcium flux were performed according to the same procedure as described recently [67 (link)]. Purified eosinophils were labeled with 3 µM Fluo-4 (Molecular Probes, Eugine, OR, USA) and stimulated with 0.1, 1, 10, or 100 µM capsaicin (Merck, Darmstadt, Germany) during the measurement. Ionomycin (500 nM) (ThermoFisher Scientific, Waltham, MA, USA) was used as the positive control and RPMI medium (VWR International, Leuven, Belgium) as the negative control. For statistical analysis, the factor of the intracellular fluorescence was calculated through comparing the intensity peak after application of stimulants at 60 s to the baseline (mean value from 40 to 45 s). This experiment was also conducted with 100 µM capsaicin after eosinophil priming with IL-3 (10 ng/mL) (PeproTech, Cranbury, NJ, USA) for 20 min at 37 °C and 5% CO₂.

DLD1 cells, a human colon cancer cell line, and its transformed counterparts were cultured and maintained as monolayers in RPMI medium (VWR International) supplemented with 10% (v/v) fetal bovine serum (Gibco, Life Technologies, Inc.) and 100 units/ml penicillin and streptomycin (Corning). The cells were incubated at 37°C with 5% CO2. Mycoplasma contamination was assessed in all cell lines using the MycoAlert PLUS mycoplasma detection kit (Lonza).

To assess CD69 externalization after TRPV1 activation, purified eosinophils were stimulated with 0.1, 1, 10, or 100 µM capsaicin (Merck, Darmstadt, Germany), with IL-3 (10 ng/mL) (PeproTech, Cranbury, NJ, USA) and GM-CSF (10 ng/mL) (BioLegend, Amsterdam, the Netherlands) as positive controls, for 24 h at 37 °C and 5% CO₂ in RPMI medium (containing 10% FCS and 1% PenStrep) (VWR International, Leuven, Belgium). To analyze CD69 and TRPV1 expression, eosinophils were stained with CD69-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and TRPV1-PE (Biozol, Eching, Germany) antibodies. FMO controls were stained without the CD69-APC or TRPV1-PE antibody, respectively. To exclude unspecific binding, cells were also stained with a PE-Rabbit Isotype Control (ab37407, Abcam, Cambridge, UK) or REA APC isotype control (Miltenyi Biotec, Bergisch Gladbach, Germany). Measurement was performed after 10 min of incubation in the dark on the CytoFlexS platform (Beckman Coulter, Brea, CA, USA). CD193-FITC, CD69-APC, CD15-PB, and CD16-APC-A750 were compensated using the MACS Comp Bead Kit anti-REA (Miltenyi Biotec, Bergisch Gladbach, Germany) for the REA antibodies and the MACS Comp Bead Kit anti-mouse Igκ (Miltenyi Biotec, Bergisch Gladbach, Germany) for the remaining antibodies, according to the manufacturer’s protocol.

Purified eosinophils were stained with annexin V and propidium iodide (Apoptosis Detection Kit, Beckman Coulter, Brea, CA, USA) to assess apoptosis after 4 and 24 h of incubation. Staurosporine (1 µM) (ThermoFisher Scientific, Waltham, MA, USA) served as the proapoptotic, IL-3 (10 ng/mL) (PeproTech, Cranbury, NJ, USA) as the anti-apoptotic, and RPMI medium (VWR International, Leuven, Belgium) as the negative control. Apoptotic stages were determined through flow cytometry.

Purified eosinophils were resuspended in RPMI medium (containing 10% and 1% PenStrep) (VWR International, Leuven, Belgium). Eosinophils were stimulated for 4 h at 37 °C and 5% CO₂ with IL-3 (10 ng/mL), IL-13 (50 ng/mL), IL-33 (10 ng/mL), TSLP (10 ng/mL), NGF-β (10 ng/mL), BDNF (50 ng/mL), TNF-α (10 ng/mL), or IL-31 (10 ng/mL) (PeproTech, Cranbury, NJ, USA). Eosinophils were also incubated without any stimulants for 4 h at 37 °C and 40 °C and at pH 5.0, 5.5, 6.0, or 7.0. The pH value of the medium was adjusted with HCl (Sigma-Aldrich, St. Louis, MO, USA) and NaOH (Carl Roth, Karlsruhe, Germany) and determined with a pH electrode.