URB 597

Immortalized HaCaT cells from the original depositor (DKFZ, Heidelberg), which were of Caucasian skin type (phototype), were purchased from CLS-Cell Lines Service (code 300493). In all of the experiments, HaCaT cells were used at the 5th–6th passage, cultured at 37 °C in a humidified 5% CO₂ atmosphere in high-glucose Dulbecco’s Modified Eagle Medium (DMEM-HG) and supplemented with a 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (pen/strep) solution. For each treatment, after 24 h of cell growth, the cell culture medium was replaced with DMEM-HG supplemented with 1% FBS and 1% pen/strep (Starvation medium) and incubated for 24 h. On the third day, the cells were incubated with the following conditions, according to the treatment of interest: control (DMEM + 5% FBS + 1% pen/strep); LPS at 1.0, 5.0 and 10.0 μg/mL; positive control Hydrocortisone (HC) at 10.0 μM; pCBs at specific pre-calculated concentrations (CBG 6.0 μM, CBC 4.0 μM, THCV 9.3 μM and CBGA 13.0 μM) [8 (link)] and the same pCBs in combination with the following selected eCB system antagonists and inhibitors: CPZ at 5.0 μM [27 (link)]; JZL184 at 10.0 μM and LEI-106 at 10.0 μM [28 (link)]; URB597 at 1.0 μM [29 (link)] and ARN19874 at 33.7 μM [30 (link)]. After 24 and 48 h, cell pellets and supernatants (medium) from each condition were collected, centrifuged at 300× g for 5 min, and stored at −20 °C and at −80 °C, respectively, after centrifugation.

HaCaT cells were seeded into 12-well plates at a density of 2 × 10⁵ per well, incubated overnight and then exposed to LPS at 5.0 µg/mL, to positive control Hydrocortisone (HC) at 10.0 μM and to selected pCBs at pre-calculated concentrations (CBG 6.0 μM, CBC 4.0 μM, THCV 9.3 μM and CBGA 13.0 μM) corresponding to half of the IC₅₀ values [8 (link)] for 24 h and 48 h. In addition, HaCaT cells were exposed to THCV and CBGA in combination with selected eCB system antagonists and inhibitors (CPZ 5.0 μM for both pCBs, JZL184 10.0 μM for THCV, LEI-106 10.0 μM for THCV, URB597 1.0 μM for CBGA and ARN19874 33.7 μM for CBGA) for 24 h in the case of IL-31 and for 48 h in the case of IL-12. After each treatment, supernatants (medium) of each condition were collected, centrifuged at 4 °C at 300× g for 5 min and stored at −80 °C until the assay. Cytokine quantification was performed using the DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) for IL-1β, IL-8, IL-10, IL-12 and IL-31, following the manufacturer’s instructions. Briefly, 96-well plates were coated overnight with the specific capture antibody for each interleukin and then blocked with 1% BSA. Standard proteins in duplicate and samples in triplicate were added and incubated for 2 h. The standard curve for each interleukin was generated by using serially diluted standard proteins at known concentrations as provided by the manufacturer, and the calibration curves obtained were used to interpolate sample values. Then, each well with standards and/or samples was incubated with the specific detection antibody for 2 h. The Streptavidin-HRP was added and incubated for 20 min, followed by the substrate solution for another 20 min, and then the reaction was stopped with the specific Stop Solution (1M H₂SO₄). As for TNF-β, quantification was performed using the Human TNF-beta Platinum ELISA 96 tests (Invitrogen, Waltham, MA, USA). Briefly, 96-well plates already coated with TNF-β capture antibody were incubated for 4 h with standard and samples along with HRP-conjugate. After this period, TMB Substrate Solution was added to all wells for 10 min of incubation and then stopped with the specific Stop Solution (1M H₃PO₄). Optical densities were determined using an Enspire microplate reader (Perkin Elmer, Waltham, MA, USA) at the specific wavelengths of 450 and 570 nm for IL-1β, IL-8, IL-10, IL-12 and IL-31 and of 450 nm and 620 nm for TNF-β.