Cell titre aqueous one solution viability assay

Cytotoxicity assays were performed as described before [17 (link)]. Briefly, L929 cells grown on 96-well plates were incubated with 20 ng/mL (1.2 nM) of hTNF or 10–20 ng/mL (1.2 nM or 2.4 nM) of mTNF for 16 h in the presence of 4 µg/mL ActinomycinD (Sigma, Saint Louis, MO, USA) to induce cell death. Relevant recombinant proteins were preincubated at the indicated doses with TNF for 2 h at 37 °C before addition to the cell monolayers. Experiments were carried out in triplicate wells. After the incubation period, cell death was assessed using the Cell Titre Aqueous One Solution viability assay (Promega, Madison, WI, USA) according to the manufacturer´s instructions. The data were analyzed for statistical significance by performing multiple t-test comparisons among selected groups using Prism 6.0 software (GraphPad, San Diego, CA, USA).

Migration of MOLT-4 cells towards chemokine was assayed using 96-well ChemoTx plates with 5 µm pores (Neuro Probe, Gaithersburg, MD, USA). Chemokine (mCCL25) at 100 nM was incubated in the presence of increasing amounts of recombinant proteins, as indicated in the bottom wells, for at least 15 min at 37 °C. Cells (2.5 × 10⁵ per well) previously washed and resuspended in 0.1% FCS in RPMI1640 medium were carefully laid on the top filter and allowed to migrate for 2–6 h at 37 °C. After this period, nonmigrated cells from the top filter were rinsed away with phosphate buffered saline (PBS), and the number of migrated cells in each well determined using the Cell Titre Aqueous One Solution viability assay (Promega, Madison, WI, USA). The number of cells was extrapolated from a standard curve with known cell numbers prepared in triplicate for each assay, as suggested by the manufacturers.