4.3.1. 2D Cell Cultures

Table 5 shows all treatments. Experiments were conducted in culture media without antibiotics and FCS (= experimental media). For gene expression analysis and ELISA, the IVD cells were seeded into T25 flasks (3.5 × 105 cells/flask) or six-well plates (3 × 105 cells/well). For immunofluorescence, <sup>1</sup> × 105 cells were seeded into the wells of chambered slides (155380, Thermo Fisher Scientific). For Calcium imaging, the 4 × 104 cells were seeded into 96-well plates in triplicates and incubated for 18 h. The next day, complete media was changed to the experimental media. After 2 h in experimental media, the cells were exposed to 5 or 10 ng/mL recombinant TNF-α (315-01A, PeproTech, Umkirch, Germany) or IL-1β (200-01B, PeproTech) for 18 h. Non-stimulated cells were used as the controls. To investigate the effects of TRPA1 and TRPV1 activation, 3 and 10 μM allyl isothiocyanate (AITC, TRPA1 agonist, 377430, Sigma) was used either in untreated cells or in IL1-β and TNF-α treated cells (focus on TRPA1, whose expression is increased in an inflammatory environment). The EC50 value for AITC that is reported in the literature is approximately 3 μM, while AITC concentrations higher than 10 μM may cause channel desensitization [32,33].
