Victor x4tm multilabel plate reader

Saliva was collected by using cortisol salivettes with a synthetic swab (Reg. Nr. 51.1534.500, Sarstedt Company, Nümbrecht, Germany). The saliva samples were stored at -20°C until shipping to the Biochemical Laboratory, Trier University, Germany, where the samples were analyzed. Saliva samples were centrifuged at 2000 g for 10 min. 100 ul of saliva were used for duplicate analysis. Cortisol levels were determined employing a competitive solid phase time-resolved fluorescence immunoassay with flouromeric end point detection (DELFIA). Standards, controls (saliva pools) and samples were given in duplicate wells. Cortisol concentrations of the samples were calculated with VICTOR X4^TM Multilabel Plate Reader (Perkin Elmer, MA, United States). The intra-assay coefficient of variation was between 4.0 and 6.7%. Inter-assay variation was eliminated by measuring all samples within the same assay [see (Dressendörfer et al., 1992 (link))].

The assays investigating differences in cell viability, apoptosis and proliferation between cellular variants were performed on a single plate. The cells were seeded in full medium on a 96-well plate at a concentration of 1.5 × 10⁴ cells per well and incubated for 24 h. The medium was then changed into 100 µL starvation medium containing 10 µL of 5-bromo-2′-deoxyuridine (BrdU) and the cells were subsequently incubated for 24 h. Following this, 10 µL PrestoBlue reagent (Thermo Fisher Scientific, Naarden, the Netherlands) was added to the wells and fluorescence (excitation at 550 nm and emission at 590 nm) were measured at 10 min intervals (control wells: without cells and reagent). Apoptosis and proliferation were detected according to the manufacturer’s protocols using TUNEL assay (DELFIA^® DNA Fragmentation Assay; PerkinElmer, Vaughan, ON, Canada) and BrdU incorporation (DELFIA^® Cell Proliferation Kit; PerkinElmer, Vaughan, ON, Canada), respectively. The VICTOR X4^TM Multilabel Plate Reader (PerkinElmer, Vaughan, ON, Canada) was used to detect fluorescence signals. The assay was performed in triplicate for each cellular variant.