Quantikine assays

A549 cells were incubated with different doses of IL-1beta and LPS in a time course. At each time point culture supernatants were harvested and the concentrations of the cytokines IL-6, IL-8, and TGF-beta were determined by Quantikine Assays (R&D Systems) according to manufacturer's instructions. ELISA was performed using Infinite M200 microplate reader (Tecan, Männedorf, Switzerland).
To determine the effects of EtOH, EtP, and NaP on the cytokine production, A549 cells were preincubated with EtOH, EtP, or NaP for 1, 24, or 72 h prior the stimulation with IL-1beta or LPS for 24 h. Then, IL-6 and IL-8 were measured in culture supernatants as described above. To uncover the differences in cytokine release in these experiments, cytokine levels in percent relative to stimulated controls are expressed.

Serum was stored at −70 °C until analysis. All assays were performed according to the instructions of the manufacturers. Calcium, phosphate, and ALP were determined using an RX Monza Analyzer (Randox, Antrim, UK). For OPG and RANKL, immunoassays were used (Quantikine assays, R&D Systems, Minneapolis, MN, USA), while OC was analyzed with an immunoradiometric assay (Immutopics, San Clemente, CA, USA). Deoxypyridinoline (DPD), a crosslink product of collagen molecules, was determined in urine samples (Quidel, San Diego, CA, USA). Creatinine was measured in urine samples within two hours after collection using a Siemens ADVIA 2400 analyzer (Siemens Medical Solutions, Fernwald, Germany). Variations in urine concentration were eliminated using the ratio of DPD [nmol/L] and the urine creatinine concentration [mmol/L], resulting in a value without units.