Cell Viability Assay with CCK-8 Kit

Assessment of cell viability was accomplished using the CCK-8 kit according to methods previously described (Hemmingsen et al., 2021b (link)). The cells (HaCaT; Cauzzo et al., 2020 (link), NHDF-neo; Domiński et al., 2022 (link), and murine macrophages RAW 264.7; Basnet et al., 2012 (link); Cauzzo et al., 2020 (link)) in complete RPMI medium [containing 10% (v/v) FBS and penicillin–streptomycin; RAW 264.7] or complete DMEM-hg (HaCaT and NHDF-neo) were plated on 96-well plates (90 μl, 1 × 10⁵ cells/ml) and incubated (37°C, 5% CO₂) for 24 h. Diluted vesicle suspensions (10 μl) were added to the wells (final lipid concentration of 1, 10, and 50 μg/ml) and the plates incubated for another 24 h (37°C, 5% CO₂). Next, an aliquot of 10 μl CCK-8 reagent was added to each well and the plates were incubated for 4 h. The cell viability was measured using Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) at 450 nm with the reference set to 650 nm. Treated cells were compared to non-treated cells (only complete RPMI or DMEM-hg).