Atplitetm m assay

For experiments, fibroblasts were harvested through trypsin-EDTA (Gibco) treatment, seeded into 12-well plates (Greiner bio-one, Germany) at a density of 10,000 cells/cm², and cultured for 24 h to 30% confluence. Afterwards, fibroblasts were incubated with the test specimen extracts (refer to sample preparation for details) for 24 to 72 h. Cells cultured in complete DMEM served as untreated control. Additionally, cells incubated with 10 ng/mL epidermal growth factor (EGF, Promocell) were used as positive control for proliferative effects. Determination of cell proliferation was carried out using the luminometric ATP assay (ATPLiteTM-M Assay, PerkinElmer). Here, the amount of viable cells was specified by cellular ATP-content in [nM]. The number of viable, metabolically active cells was further determined using the photometric MTT assay (MTT Cell Proliferation Assay Kit, Invitrogen). The cell amounts are given as the absorbance measured at 580 nm in [mOD].

Cell adherence to material surface of samples removed from the NHDF monolayer after 1, 24, and 48 h from the previous experiment (see “Determination of the effect on cell viability”) was also investigated. Cell presence was ascertained using a luminometric adenosine triphosphate (ATP) assay (ATPLite^TM-M Assay, PerkinElmer) that determines the cellular ATP concentration based on light generated during the ATP-dependent conversion of luciferin by luciferase. Luminescence was measured using a microplate luminometer (LUMIstar Galaxy, BMG Labtech, Germany). The ATP concentrations were calculated based on a standard curve and the number of viable cells specified as cellular ATP-content in [nM].