Epoch plate spectrophotometer

We determined the acid and pepsin soluble collagen on fibroblast supernatants using the Sircol assay (Biocolor Antrim, UK). 1-1.5 × 10⁶ cells seeded on 75 cm² flask were used by condition. Cells were stimulated for 48 hrs with 100 ng/ml of CX3CL1 (Biolegend San Diego, USA); non-stimulated cells were also tested. After stimulation, culture medium was collected, lyophilized and reconstituted in 70 µl deionized water. The concentration of collagen in 50 µl of each sample was evaluated by the Sircol assay according to the manufacturer's instructions; each sample was run in duplicates. Samples were read at OD₅₅₀ nm and at OD₆₀₀ nm in an Epoch plate spectrophotometer (Biotek, Winooski, USA). Concentration of collagen was calculated with a standard curve using the Gen 5 software (Biotek).

To determine the cytotoxic and antiproliferative effect of c9, t11 CLA on examined cell lines we used MTT proliferation assay (Sigma Aldrich, St. Louis, USA). Cells were cultured in DMEM/F12 (Beas-2B) or RPMI 1640 (A549, Calu-1) medium enriched with 10% FBS for 24 h and then seeded into 96-well plates at the density of 3.5 × 10³ (A549 and Beas-2B) and 7.0 × 10³ (Calu-1) cells per well. Next, cells were incubated in the presence of different doses of CLA (25 μM, 50 μM, 75 μM for Beas-2B and Calu-1; 50 μM, 100 μM, 200 μM for A549) for 24, 48 and 72 h. After the incubation period, all media were removed and replaced by 200 μl of 10% MTT solution (5 mg of MTT substrate per 1 ml of PBS) in FBS-free cell culture medium and left for 4 h in the incubator. After this time MTT mixture was carefully discarded and newly formatted formazan crystals were dissolved by adding 50 μl of DMSO into each well. The absorbance was measured by Epoch Plate Spectrophotometer (BioTek, Vinooski,VT, USA). Each experiment was performed in 8 biological repeats for each CLA concentration and included 8 vehicle controls (with DMSO).