Sirius ht

HPK were seeded in a 96-well plate (6000 cells per well) over night and were incubated with basal Keratinocyte-SFM medium (Thermo Fisher, Darmstadt, Germany) for 24 h. Then, the cells were incubated for a further 24 h with the plant extracts before the cell viability was assessed with the CellTiter-Glo2.0 Assay (Promega, Walldorf, Germany) according to the manufacturer’s protocol. The method is based on the bioluminescent measurement of ATP that is present in metabolically active cells. Luciferase catalyzes the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration and is measured using a scanning multiwell spectrophotometer (Sirius HT from BioTek, Bad Friedrichshall, Germany).

Proliferation was assessed by the BrdU cell proliferation assay (Roche, Mannheim, Germany). 2 × 10³ HPK were seeded per well in a 96-well plate and incubated for 24 h at 37 °C under 5% CO₂. Then, the cells were incubated with basal Keratinocyte-SFM medium (Thermo Fisher, Darmstadt, Germany) for 24 h and stimulated for 72 h with psoriasis cytokines (IL-17A, IL-22 and TNF-α, 20 ng/mL each) before the plant extracts were added for 24 h. The BrdU assay labeling occurred according to the manufacturer’s instruction. Absorption at a wavelength of 450 nm was measured in a scanning multiwell spectrophotometer (Sirius HT from BioTek, Bad Friedrichshall, Germany).

The lipid content in the differentiated 3T3-L1 adipocytes was determined using the Oil Red O staining method, according to the method published by Kowalska et al. [21 (link)] with slight modifications. Briefly, the cells were washed with PBS after incubation, fixed in 250 µL formalin (10%) for 1 h, and washed with 60% isopropanol. The cells were then incubated with 100 µL Oil red O staining solution (0.21% in 60% isopropanol) for 10 min at room temperature, washed with water to remove unbound dye, and left to dry. Fat droplets, stained red, were extracted from cells using 375 µL 100% isopropanol, and the absorbance was measured at a wavelength of 520 nm with a microplate reader (Sirius HT, BioTek Instruments GmbH, Bad Friedrichshall, Germany). Resveratrol (c = 11.4 µg/mL and 22.8 µg/mL) was used as a positive control, according to Fischer-Posovsky et al. [26 (link)]. Lipid accumulation is presented as the percentage of the solvent control (DMSO; final concentration 0.1%).