Inflammatory Response in HaCaT Cells: Modulation by Phytocannabinoids

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.