Crosslinker blx 312

When KCs formed all derived lines reached 70% confluence, cells were exposed to UVB (312 nm) radiation in cold PBS (4°C) to avoid heat stress and oxidation of the medium components. Used wavelength corresponds to the wavelength of the UVB radiation (Narrov Band) used in the phototherapy of psoriasis [22 (link)]. KCs were irradiated on ice at a distance of 15 cm from an assembly of 6 UV lamps (Bio-Link Crosslinker BLX 312; Vilber Lourmat, Germany) at 6 W each, corresponding to a flux of 4.08 mW/cm². Total UVB dose was 60 mJ/cm², which resulted in the death of approximately 30% of KCs. After irradiation, KCs were incubated for 24 hours under standard conditions in medium without supplementation with growth factors. To analyze the effect of CBD on these cells, a suspension of CBD in ethanol was added to a final concentration of 4 μM (the final concentration of ethanol was 0.3%). Following incubation (24 h), the medium from was harvested and frozen at -80°C before further analysis.

Human keratinocytes (CDD 1102 KERTr) obtained from American Type Culture Collection (ATCC, Virginia, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), together with 10% fetal bovine serum (FBS) supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C. After keratinocytes were grown to 70% confluence, they were passaged at a ratio of 1:3. After reaching the required confluency for all passages, keratinocytes were divided into two main cell groups as described below:
Control groups of keratinocytes

Control cells cultured with standard medium (CTR).

Cells cultured in medium containing 4 µM CBD (Sigma-Aldrich, MO, USA—98.5% pure) in 0.2% ethanol for 48 h (CBD(48 h))

Cells cultured in medium containing 4 µM CBD in 0.2% ethanol for 24 h (CBD(24 h)).

UVB-irradiated groups of keratinocytes
IV.

Cells irradiated by UVB (312 nm) at 60 mJ/cm² (Bio-Link Crosslinker BLX 312; Vilber Lourmat, Germany; six lamps at a distance of 15 cm) (UVB).

V.

Cells cultured in medium containing 4 μM CBD in 0.2% ethanol for 24 h before and 24 h after UVB irradiation (pretreatment + treatment) (CBD + UVB + CBD)

VI.

Cells cultured in medium containing 4 μM CBD in 0.2% ethanol for 24 h after UVB irradiation (treatment only) (UVB + CBD).